There is a dominant view that plants are shaped by the ability of their outermost cell layers to resist or yield to pressure from internal tissues. During stem development, outer tissues originate from the tunica layers of the shoot meristem, while inner tissues are produced by the rib zone (RZ), named after its distinctive pattern of transverse cell divisions. Despite over a century of studies, the interplay between inner and outer tissues in shaping the stem remains unclear. Here, we show that mutations in a subfamily of IQ domain (IQD) genes disrupt the orientation of cell divisions in the RZ and increase stem diameter in Arabidopsis. Measurements of cell geometry, growth of marked cell clones, tissue-specific expression, and subcellular localization all support the idea that these IQD proteins reduce longitudinal cell divisions in the RZ, limiting the build-up of vertical cell files in inner tissues and the concomitant radial growth of the stem. Thus, the characteristic orientation of cell divisions in the RZ is important for shaping the stem, and the genetic control of organ shape can be exerted in inner plant tissues.

The plant vasculature plays essential roles in the transport of water and nutrients and is composed of xylem and phloem, both of which originate from undifferentiated cells found in the cambium. Development of the different vascular tissues is coordinated by hormonal and peptide signals and culminates in extensive cell wall modifications. Pectins are key cell wall components that are modified during cell growth and differentiation, and pectin fragments function as signals in defence and cell wall integrity pathways, although their role as developmental signals remains tentative. Here, we show that the pectin lyase-like gene PLL12 is required for growth of the vascular bundles in the Arabidopsis inflorescence stem. Although PLL12 was expressed primarily in the phloem, it also affected cambium and xylem growth. Surprisingly, PLL12 overexpression induced ectopic cambium and xylem differentiation in the inflorescence apex and inhibited development of the leaf vasculature. Our results raise the possibility that a cell wall-derived signal produced by PLL12 in the phloem regulates cambium and xylem development.

The diversity and environmental plasticity of plant growth results from variations of repetitive modules, such as the basic shoot units made of a leaf, axillary bud, and internode. Internode elongation is regulated both developmentally and in response to environmental conditions, such as light quality, but the integration of internal and environmental signals is poorly understood. Here, we show that the compressed rosette growth habit of Arabidopsis is maintained by the convergent activities of the organ boundary gene ARABIDOPSIS THALIANA HOMEOBOX GENE 1 (ATH1) and of the gibberellin-signaling DELLA genes. Combined loss of ATH1 and DELLA function activated stem development during the vegetative phase and changed the growth habit from rosette to caulescent. Chromatin immunoprecipitation high-throughput sequencing and genetic analysis indicated that ATH1 and the DELLA gene REPRESSOR OF GA1-3 (RGA) converge on the regulation of light responses, including the PHYTOCHROME INTERACTING FACTORS (PIF) pathway, and showed that the ATH1 input is mediated in part by direct activation of BLADE ON PETIOLE (BOP1 and BOP2) genes, whose products destabilize PIF proteins. We conclude that an organ-patterning gene converges with hormone signaling to spatially restrict environmental responses and establish a widespread type of plant architecture.

Ploidy and size phenomena are observed to be correlated across several biological scales, from subcellular to organismal. Two kinds of ploidy change can affect plants. Whole-genome multiplication increases ploidy in whole plants and is broadly associated with increases in cell and organism size. Endoreduplication increases ploidy in individual cells. Ploidy increase is strongly correlated with increased cell size and nuclear volume. Here, we investigate scaling relationships between ploidy and size by simultaneously quantifying nuclear size, cell size, and organ size in sepals from an isogenic series of diploid, tetraploid, and octoploid Arabidopsis thaliana plants, each of which contains an internal endopolyploidy series. We find that pavement cell size and transcriptome size increase linearly with whole-organism ploidy, but organ area increases more modestly due to a compensatory decrease in cell number. We observe that cell size and nuclear size are maintained at a constant ratio; the value of this constant is similar in diploid and tetraploid plants and slightly lower in octoploid plants. However, cell size is maintained in a mutant with reduced nuclear size, indicating that cell size is scaled to cell ploidy rather than to nuclear size. These results shed light on how size is regulated in plants and how cells and organisms of differing sizes are generated by ploidy change.

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 regulators(3). 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 Sln1d(4) 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.

Across 13 experiments the gw2 - A1 mutant allele shifts grain size distribution consistently across all grains significantly increasing grain weight (6.6 %), width (2.8 %) and length (2.1 %) in tetraploid and hexaploid wheat. There is an urgent need to identify, understand and incorporate alleles that benefit yield in polyploid wheat. The rice OsGW2 gene functions as a negative regulator of grain weight and width and is homologous to the wheat TaGW2 gene. Previously it was shown that transcript levels of the A-genome homoeologue, TaGW2-A1, are negatively associated with grain width in hexaploid wheat. In this study we screened the tetraploid Kronos TILLING population to identify mutants in TaGW2-A1. We identified a G to A transition in the splice acceptor site of exon 5 which leads to mis-splicing in TaGW2-A1. We backcrossed the mutant allele into tetraploid and hexaploid wheat and generated a series of backcross derived isogenic lines which were evaluated in glasshouse and field conditions. Across 13 experiments the GW2-A1 mutant allele significantly increased thousand grain weight (6.6 %), grain width (2.8 %) and grain length (2.1 %) in tetraploid and hexaploid wheat compared to the wild type allele. In hexaploid wheat, this led to an increase in spike yield since no differences were detected for spikelet or grain number between isogenic lines. The increase in grain width and length was consistent across grains of different sizes, suggesting that the effect of the mutation is stable across the ear and within spikelets. Differences in carpel size and weight between alleles were identified as early as 5 days before anthesis, suggesting that TaGW2-A1 acts on maternal tissue before anthesis to restrict seed size. A single nucleotide polymorphism marker was developed to aid the deployment of the mutant allele into breeding programmes.

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.

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.

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.

This chapter contains sections titled: Protein Compartmentalization in Plant Cells Experimental Determination of Protein Localization In Vivo Imaging Approaches to Protein Localization and Interaction Plant Cell Cultures for Studying Protein Localization Protein–Protein Interaction In Vivo: FRET Perspectives: Integrating Predictive and Experimental Protein Localization Data References

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.

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.

The Aspergillus alc regulon encodes a transcription factor, ALCR, which regulates transcription from cognate promoters such as alcA(p). In the presence of suitable chemical inducers, ALCR activates gene expression from alcA(p). The alc regulon can be transferred to other species and can be used to control the expression of reporter, metabolic and developmental genes in response to low-level ethanol exposure. In this paper, we describe a versatile system for targeting the alc regulon to specific cell types in Arabidopsis by driving ALCR expression from the GAL4 upstream activator sequence (UAS). Large numbers of Arabidopsis lines are available in which GAL4 is expressed in a variety of spatial patterns and, in turn, drives the expression of any gene cloned downstream of the UAS. We have used a previously characterized line that directs gene expression to the endosperm to demonstrate spatially restricted ethanol-inducible gene expression. We also show that the domain of inducible gene expression can easily be altered by crossing the UAS::ALCR cassette into different driver lines. We conclude that this gene switch can be used to drive gene expression in a highly responsive, but spatially restricted, manner.

Aquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS-PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So-deltaTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity-purified So-deltaTIP-specific peptide antibodies, we investigated the subcellular and tissue distribution of So-deltaTIP. So-deltaTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between delta-TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So-deltaTIP with that of more distantly related TIPs showed similarities with Arabidopsis gamma-TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So-deltaTIP may be induced when the large vacuole is formed.