Max Bush

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

Arabidopsis POT1A interacts with TERT-V(I8), an N-terminal splicing variant of telomerase

Chromosome integrity is maintained via the actions of ribonucleoprotein complexes that can add telomeric repeats or can protect the chromosome end from being degraded. POT1 (protection of telomeres 1), a class of single-stranded-DNA-binding proteins, is a regulator of telomeric length. The Arabidopsis genome contains three POT1 homologues: POT1A, POT1B and POT1C. Using yeast two-hybrid assays to identify components of a potential POT1A complex, we retrieved three interactors: the N-terminus of the telomerase, a protein kinase and a plant-specific protein. Further analysis of the interaction of POT1 proteins with telomerase showed that this interaction is specific to POT1A, suggesting a specific role for this paralogue. The interaction is specific to the N-terminal region of the telomerase, which can be encoded by splicing variants. This interaction indicates possible mechanisms for telomerase regulation by alternative splicing and by POT1 proteins.

JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis

Summary Background In all multicellular organisms, the links between patterning genes, cell growth, cell cycle, cell size homeostasis, and organ growth are poorly understood, partly due to the difficulty of dynamic, 3D analysis of cell behavior in growing organs. A crucial step in plant organogenesis is the emergence of organ primordia from the apical meristems. Here, we combined quantitative, 3D analysis of cell geometry and DNA synthesis to study the role of the transcription factor JAGGED (JAG), which functions at the interface between patterning and primordium growth in Arabidopsis flowers. Results The floral meristem showed isotropic growth and tight coordination between cell volume and DNA synthesis. Sepal primordia had accelerated cell division, cell enlargement, anisotropic growth, and decoupling of DNA synthesis from cell volume, with a concomitant increase in cell size heterogeneity. All these changes in growth parameters required JAG and were genetically separable from primordium emergence. Ectopic JAG activity in the meristem promoted entry into S phase at inappropriately small cell volumes, suggesting that JAG can override a cell size checkpoint that operates in the meristem. Consistent with a role in the transition from meristem to primordium identity, JAG directly repressed the meristem regulatory genes BREVIPEDICELLUS and BELL 1 in developing flowers. Conclusions We define the cellular basis for the transition from meristem to organ identity and identify JAG as a key regulator of this transition. JAG promotes anisotropic growth and is required for changes in cell size homeostasis associated with accelerated growth and the onset of differentiation in organ primordia.

A cyclin-dependent protein kinase, CDKC2, colocalizes with and modulates the distribution of spliceosomal components in Arabidopsis

Cyclin-dependent kinases (CDKs) play key regulatory roles in diverse cellular functions, including cell-cycle progression, transcription and translation. In plants, CDKs have been classified into several groups, named A through to G, but the functions of most are poorly characterized. CDKCs are known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAP II), and therefore the CDKC-cyclinT (CycT) complex may have a role similar to the animal CDK9-CycT complex of the positive transcription elongation factor b (P-TEFb). However, we found that the predicted structure of the Arabidopsis CDKC2 protein is more similar to the mammalian cdc2-related kinase, CRK7, than to CDK9. CRK7 is proposed to link transcription with splicing, and CDKC2 contains all the structural features of CRK7 that make the latter distinct from CDK9. Consistent with this, we show that GFP-CDKC2 fusion proteins co-localize with spliceosomal components, that the expression of CDKC2 modifies the location of these components, and that co-localization was dependent on the transcriptional status of the cells and on CDKC2-kinase activity. We propose, therefore, that the Arabidopsis CDKC2 combines the functions of both CRK7 and CDK9, and could also couple splicing with transcription.

A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon

In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.

Control of Oriented Tissue Growth through Repression of Organ Boundary Genes Promotes Stem Morphogenesis

Summary The origin of the stem is a major but poorly understood aspect of plant development, partly because the stem initiates in a relatively inaccessible region of the shoot apical meristem called the rib zone (RZ). We developed quantitative 3D image analysis and clonal analysis tools, which revealed that the Arabidopsis homeodomain protein REPLUMLESS (RPL) establishes distinct patterns of oriented cell division and growth in the central and peripheral regions of the RZ. A genome-wide screen for target genes connected RPL directly to many of the key shoot development pathways, including the development of organ boundaries; accordingly, mutation of the organ boundary gene LIGHT-SENSITIVE HYPOCOTYL 4 restored RZ function and stem growth in the rpl mutant. Our work opens the way to study a developmental process of importance to crop improvement and highlights how apparently simple changes in 3D organ growth can reflect more complex internal changes in oriented cell activities.

Remodelling pectin structure in potato

Figure options Download full-size image Download as PowerPoint slide Pectin is a collection of polysaccharides, which play an important role in controlling the pore size of the plant cell wall, regulating cell-cell adhesion, and providing a source of sig-nalling molecules that elicit a range of cellular responses. Apart from this, pectins are of interest because they are an attractive hydrocolloid for various food applications. The kind and distribution of decorative groups in the pectic molecules largely determines for which application a particular pectin is most suitable. After the extraction of starch from potato tubers, a by-product is obtained, which is relatively rich in pectin. However, the quality of these pectins is poor compared to that from other sources such as citrus and apple. Rather than trying to change the structural characteristics of potato pectin post-harvest, we have embarked on achieving this in the potato plant itself. This paper summarises the structural features of pectin, the distribution of various pectic epitopes in tuber cell walls, the enzymes involved in its biosynthesis and degradation, and strategies employed to alter its fine structure in planta.

DELLA genes restrict inflorescence meristem function independently of plant height

DELLA proteins associate with transcription factors to control plant growth in response to gibberellin1. Semi-dwarf DELLA mutants with improved harvest index and decreased lodging greatly improved global food security during the ‘green revolution’ in the 1960–1970s2. However, DELLA mutants are pleiotropic and the developmental basis for their effects on plant architecture remains poorly understood. Here, we show that DELLA proteins have genetically separable roles in controlling stem growth and the size of the inflorescence meristem, where flowers initiate. Quantitative three-dimensional image analysis, combined with a genome-wide screen for DELLA-bound loci in the inflorescence tip, revealed that DELLAs limit meristem size in Arabidopsis by directly upregulating the cell-cycle inhibitor KRP2 in the underlying rib meristem, without affecting the canonical WUSCHEL-CLAVATA meristem size regulators3. Mutation of KRP2 in a DELLA semi-dwarf background restored meristem size, but not stem growth, and accelerated flower production. In barley, secondary mutations in the DELLA gain-of-function mutant Sln1d4 also uncoupled meristem and inflorescence size from plant height. Our work reveals an unexpected and conserved role for DELLA genes in controlling shoot meristem function and suggests how dissection of pleiotropic DELLA functions could unlock further yield gains in semi-dwarf mutants.During gibberellic acid signalling, DELLAs restrict the size of the shoot apical meristem by targeting the cell cycle regulator KRP2. The roles of DELLAs in the shoot apical meristem and stem growth can be genetically uncoupled in Arabidopsis and barley.