Bimodular auxin response controls organogenesis in Arabidopsis.

Like animals, the mature plant body develops via successive sets of instructions that determine cell fate, patterning, and organogenesis. In the coordination of various developmental programs, several plant hormones play decisive roles, among which auxin is the best-documented hormonal signal. Despite the broad range of processes influenced by auxin, how such a single signaling molecule can be translated into a multitude of distinct responses remains unclear. In Arabidopsis thaliana, lateral root development is a classic example of a developmental process that is controlled by auxin at multiple stages. Therefore, we used lateral root formation as a model system to gain insight into the multifunctionality of auxin. We were able to demonstrate the complementary and sequential action of two discrete auxin response modules, the previously described Solitary Root/indole-3-Acetic Acid (IAA)14-Auxin Response Factor (ARF)7-ARF19-dependent lateral root initiation module and the successive Bodenlos/IAA12-Monopteros/ARF5-dependent module, both of which are required for proper organogenesis. The genetic framework in which two successive auxin response modules control early steps of a developmental process adds an extra dimension to the complexity of auxin's action.

Gene silencing induced by hairpin or inverted repeated sense transgenes varies among promoters and cell types.

*In transgenic calli and different tissues of Arabidopsis thaliana plants, the in trans silencing capacity of a 35S-beta-glucuronidase (GUS) hairpin RNA construct was investigated on a target GUS gene, under the control of the 35S, a WRKY or several cell cycle-specific promoters. *GUS histochemical staining patterns were analyzed in all tissues of the parental lines and supertransformants harboring the hairpin construct. Quantitative GUS activity measurements determined GUS suppression by a 35S-GUS hairpin or inverted repeated GUS transgenes in leaves and calli. *In some supertransformants, GUS-based staining disappeared in all tissues, including calli. In most supertransformants, however, a significant reduction was found in mature roots and leaves, but residual GUS activity was observed in the root tips, young leaves and calli. In leaves of most hairpin RNA supertransformants, the GUS activity was reduced by c. 1000-fold or more, but, in derived calli, generally by less than 200-fold. The silencing efficiency of inverted repeated sense transgenes was similar to that of a hairpin RNA construct in leaves, but weaker in calli. *These results imply that the tissue type, nature of the silencing inducer locus and the differential expression of the targeted gene codetermine the silencing efficiency.

Extensive expression regulation and lack of heterologous enzymatic activity of the Class II trehalose metabolism proteins from Arabidopsis thaliana.

Trehalose metabolism has profound effects on plant growth and metabolism, but the mechanisms involved are unclear. In Arabidopsis, 21 putative trehalose biosynthesis genes are classified in three subfamilies based on their similarity with yeast TPS1 (encoding a trehalose-6-phosphate synthase, TPS) or TPS2 (encoding a trehalose-6-phosphate phosphatase, TPP). Although TPS1 (Class I) and TPPA and TPPB (Class III) proteins have established TPS and TPP activity, respectively, the function of the Class II proteins (AtTPS5-AtTPS11) remains elusive. A complete set of promoter-beta-glucurinidase/green fluorescent protein reporters demonstrates their remarkably differential tissue-specific expression and responsiveness to carbon availability and hormones. Heterologous expression in yeast furthermore suggests that none of the encoded enzymes displays significant TPS or TPP activity, consistent with a regulatory rather than metabolic function for this remarkable class of proteins.

Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root.

During the development of multicellular organisms, organogenesis and pattern formation depend on formative divisions to specify and maintain pools of stem cells. In higher plants, these activities are essential to shape the final root architecture because the functioning of root apical meristems and the de novo formation of lateral roots entirely rely on it. We used transcript profiling on sorted pericycle cells undergoing lateral root initiation to identify the receptor-like kinase ACR4 of Arabidopsis as a key factor both in promoting formative cell divisions in the pericycle and in constraining the number of these divisions once organogenesis has been started. In the root tip meristem, ACR4 shows a similar action by controlling cell proliferation activity in the columella cell lineage. Thus, ACR4 function reveals a common mechanism of formative cell division control in the main root tip meristem and during lateral root initiation.

Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis.

In plants, the developmental mechanisms that regulate the positioning of lateral organs along the primary root are currently unknown. We present evidence on how lateral root initiation is controlled in a spatiotemporal manner in the model plant Arabidopsis thaliana. First, lateral roots are spaced along the main axis in a regular left-right alternating pattern that correlates with gravity-induced waving and depends on AUX1, an auxin influx carrier essential for gravitropic response. Second, we found evidence that the priming of pericycle cells for lateral root initiation might take place in the basal meristem, correlating with elevated auxin sensitivity in this part of the root. This local auxin responsiveness oscillates with peaks of expression at regular intervals of 15 hours. Each peak in the auxin-reporter maximum correlates with the formation of a consecutive lateral root. Third, auxin signaling in the basal meristem triggers pericycle cells for lateral root initiation prior to the action of INDOLE-3-ACETIC ACID14 (SOLITARY ROOT).

Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana.

To study the mechanisms behind auxin-induced cell division, lateral root initiation was used as a model system. By means of microarray analysis, genome-wide transcriptional changes were monitored during the early steps of lateral root initiation. Inclusion of the dominant auxin signaling mutant solitary root1 (slr1) identified genes involved in lateral root initiation that act downstream of the auxin/indole-3-acetic acid (AUX/IAA) signaling pathway. Interestingly, key components of the cell cycle machinery were strongly defective in slr1, suggesting a direct link between AUX/IAA signaling and core cell cycle regulation. However, induction of the cell cycle in the mutant background by overexpression of the D-type cyclin (CYCD3;1) was able to trigger complete rounds of cell division in the pericycle that did not result in lateral root formation. Therefore, lateral root initiation can only take place when cell cycle activation is accompanied by cell fate respecification of pericycle cells. The microarray data also yielded evidence for the existence of both negative and positive feedback mechanisms that regulate auxin homeostasis and signal transduction in the pericycle, thereby fine-tuning the process of lateral root initiation.

B1-type cyclin-dependent kinases are essential for the formation of stomatal complexes in Arabidopsis thaliana.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. In yeasts, only one CDK is sufficient to drive cells through the cell cycle, whereas higher eukaryotes developed a family of related CDKs. Curiously, plants contain a unique class of CDKs (B-type CDKs), whose function is still unclear. We show that the CDKB1;1 gene of Arabidopsis (Arabidopsis thaliana) is highly expressed in guard cells and stomatal precursor cells of cotyledons, suggesting a prominent role for B-type CDKs in stomatal development. In accordance, transgenic Arabidopsis plants with reduced B-type CDK activity had a decreased stomatal index because of an early block of meristemoid division and inhibition of satellite meristemoid formation. Many aberrant stomatal cells were observed, all of them blocked in the G2 phase of the cell cycle. Although division of stomatal precursors was inhibited, cells still acquired stomatal identity, illustrating that stomatal cell differentiation is independent of cellular and nuclear division.

Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor.

New plant cells arise at the meristems, where they divide a few times before they leave the cell-cycle program and start to differentiate. Here we show that the E2Fa-DPa transcription factor of Arabidopsis thaliana is a key regulator determining the proliferative status of plant cells. Ectopic expression of E2Fa induced sustained cell proliferation in normally differentiated cotyledon and hypocotyl cells. The phenotype was enhanced strongly by the co-expression of E2Fa with its dimerization partner, DPa. In endoreduplicating cells, E2Fa--DPa also caused extra DNA replication that was correlated with transcriptional induction of S phase genes. Because E2Fa--DPa transgenic plants arrested early in development, we argue that controlled exit of the cell cycle is a prerequisite for normal plant development.