PERK-KIPK-KCBP signalling negatively regulates root growth in Arabidopsis thaliana.

The Arabidopsis proline-rich, extensin-like receptor-like kinases (PERKs) are a small group of receptor-like kinases that are thought to act as sensors at the cell wall through their predicted proline-rich extracellular domains. In this study, we focused on the characterization of a subclade of three Arabidopsis predicted PERK genes, PERK8, -9, and -10, for which no functions were known. Yeast two-hybrid interaction studies were conducted with the PERK8,- 9, and -10 cytosolic kinase domains, and two members of the Arabidopsis AGC VIII kinase family were identified as interacting proteins: AGC1-9 and the closely related kinesin-like calmodulin-binding protein (KCBP)-interacting protein kinase (KIPK). As KIPK has been identified previously as an interactor of KCBP, these interactions were also examined further and confirmed in this study. Finally, T-DNA mutants for each gene were screened for altered phenotypes under different conditions, and from these screens, a role for the PERK, KIPK, and KCBP genes in negatively regulating root growth was uncovered.

The FRIABLE1 gene product affects cell adhesion in Arabidopsis.

Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1), was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246). Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.

HUA2 caused natural variation in shoot morphology of A. thaliana.

Differences in life-history strategy are thought to contribute to adaptation to specific environmental conditions. Among life-history traits in plants, flowering time and shoot morphology are particularly important for reproductive success. Even though flowering time and shoot morphology are linked, the evolutionary changes in the genetic circuitry that simultaneously affects both traits remain obscure. Here, we have identified changes in a putative pre-mRNA processing factor, HUA2, as being responsible for the distinct shoot morphology and flowering behavior in Sy-0, a natural strain of Arabidopsis. HUA2 has previously been shown to positively regulate two MADS box genes affecting flowering time (FLOWERING LOCUS C [FLC]) and floral patterning (AGAMOUS [AG]) [1, 2]. We demonstrate that natural changes in HUA2 activity have opposite effects on its known functions, thus having implications for the coordinate control of induction and maintenance of floral fate. The changes in Sy-0 lead to enhanced FLC expression, resulting in an enlarged basal rosette and aerial rosettes, whereas suppression of AG function favors a reversion of floral meristems from determinate to indeterminate development. Natural variation in HUA2 activity thus coordinates changes in two important life-history traits, flowering time and shoot morphology.

Sentinels at the wall: cell wall receptors and sensors.

The emerging view of the plant cell wall is of a dynamic and responsive structure that exists as part of a continuum with the plasma membrane and cytoskeleton. This continuum must be responsive and adaptable to normal processes of growth as well as to stresses such as wounding, attack from pathogens and mechanical stimuli. Cell expansion involving wall loosening, deposition of new materials, and subsequent rigidification must be tightly regulated to allow the maintenance of cell wall integrity and co-ordination of development. Similarly, sensing and feedback are necessary for the plant to respond to mechanical stress or pathogen attack. Currently, understanding of the sensing and feedback mechanisms utilized by plants to regulate these processes is limited, although we can learn from yeast, where the signalling pathways have been more clearly defined. Plant cell walls possess a unique and complicated structure, but it is the protein components of the wall that are likely to play a crucial role at the forefront of perception, and these are likely to include a variety of sensor and receptor systems. Recent plant research has yielded a number of interesting candidates for cell wall sensors and receptors, and we are beginning to understand the role that they may play in this crucial aspect of plant biology.

Spatial organisation of four enzymes from Stevia rebaudiana that are involved in steviol glycoside synthesis.

The sweet steviol glycosides found in the leaves of Stevia rebaudiana Bert. are derived from the diterpene steviol which is produced from a branch of the gibberellic acid (GA) biosynthetic pathway. An understanding of the spatial organisation of the two pathways including subcellular compartmentation provides important insight for the metabolic engineering of steviol glycosides as well as other secondary metabolites in plants. The final step of GA biosynthesis, before the branch point for steviol production, is the formation of (-)-kaurenoic acid from (-)-kaurene, catalysed by kaurene oxidase (KO). Downstream of this, the first committed step in steviol glycoside synthesis is the hydroxylation of kaurenoic acid to form steviol which is then sequentially glucosylated by a series of UDP-glucosyltransferases (UGTs) to produce the variety of steviol glycosides. The subcellular location of KO and three of the UGTs involved in steviol glycoside biosynthesis was investigated by expression of GFP fusions and cell fractionation which revealed KO to be associated with the endoplasmic reticulum and the UGTs in the cytoplasm. It has also been shown by expressing the Stevia UGTs in Arabidopsis that the pathway can be partially reconstituted by recruitment of a native Arabidopsis glucosyltransferase.

Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana.

Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.

The synergistic activation of FLOWERING LOCUS C by FRIGIDA and a new flowering gene AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis.

The genetic changes underlying the diversification of plant forms represent a key question in understanding plant macroevolution. To understand the mechanisms leading to novel plant morphologies we investigated the Sy-0 ecotype of Arabidopsis that forms an enlarged basal rosette of leaves, develops aerial rosettes in the axils of cauline leaves, and exhibits inflorescence and floral reversion. Here we show that this heterochronic shift in reproductive development of all shoot meristems requires interaction between dominant alleles at AERIAL ROSETTE 1 (ART1), FRIGIDA (FRI), and FLOWERING LOCUS C (FLC) loci. ART1 is a new flowering gene that maps 14 cM proximal to FLC on chromosome V. ART1 activates FLC expression through a novel flowering pathway that is independent of FRI and independent of the autonomous and vernalization pathways. Synergistic activation of the floral repressor FLC by ART1 and FRI is required for delayed onset of reproductive development of all shoot meristems, leading to the Sy-0 phenotype. These results demonstrate that modulation in flowering-time genes is one of the mechanisms leading to morphological novelties.