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Plants respond to day/night cycling in a number of physiological ways. At the mRNA level, the expression of some genes changes during the 24-hr period. To identify novel genes regulated in this way, we used microarrays containing 11,521 Arabidopsis expressed sequence tags, representing an estimated 7800 unique genes, to determine gene expression levels at 6-hr intervals throughout the day. Eleven percent of the genes, encompassing genes expressed at both high and low levels, showed a diurnal expression pattern. Approximately 2% cycled with a circadian rhythm. By clustering microarray data from 47 additional nonrelated experiments, we identified groups of genes regulated only by the circadian clock. These groups contained the already characterized clock-associated genes LHY, CCA1, and GI, suggesting that other key circadian clock genes might be found within these clusters.

ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected in athb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.

A collection of 8,000 Arabidopsis thaliana plants carrying 48,000 insertions of the maize transposable element En-1 has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes. En-1 showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases, En-1 was inserted within a few kilobases upstream or down-stream of the gene. En-1 was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained an En-1 insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containing En-1 insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated that TRANSPARENT TESTA 6 (TT6) encodes flavanone 3-hydroxylase. The f3h and fls null mutants complete the set of A. thaliana lines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence

Carpels are essential for sexual plant reproduction because they house the ovules and subsequently develop into fruits that protect, nourish and ultimately disperse the seeds. The AGAMOUS ( AG ) gene is necessary for plant sexual reproduction because stamens and carpels are absent from ag mutant flowers 1 , 2 . However, the fact that sepals are converted into carpelloid organs in certain mutant backgrounds even in the absence of AG activity indicates that an AG -independent carpel-development pathway exists 2 . AG is a member of a monophyletic clade of MADS-box genes that includes SHATTERPROOF1 ( SHP1 ), SHP2 and SEEDSTICK ( STK ) 3 , indicating that these four genes might share partly redundant activities. Here we show that the SHP genes are responsible for AG -independent carpel development. We also show that the STK gene is required for normal development of the funiculus, an umbilical-cord-like structure that connects the developing seed to the fruit, and for dispersal of the seeds when the fruit matures. We further show that all four members of the AG clade are required for specifying the identity of ovules, the landmark invention during the course of vascular plant evolution that enabled seed plants to become the most successful group of land plants 4 .

Abnormal flowers have been recognized for thousands of years, but only in the past decade have the mysteries of flower development begun to unfold. Among these mysteries is the differentiation of four distinct organ types (sepals, petals, stamens and carpels), each of which may be a modified leaf 1 . A landmark accomplishment in plant developmental biology is the ABC model of flower organ identity 2 , 3 . This simple model provides a conceptual framework for explaining how the individual and combined activities of the ABC genes produce the four organ types of the typical eudicot flower. Here we show that the activities of the B  and C organ-identity genes require the activities of three closely related and functionally redundant MADS-box genes, SEPALLATA1/2/3 (SEP1/2/3) . Triple mutant Arabidopsis plants lacking the activity of all three SEP genes produce flowers in which all organs develop as sepals. Thus SEP1/2/3 are a class of organ-identity genes that is required for development of petals, stamens and carpels.

The behavior of the autonomous maize transposable element En/Spm of maize was studied in Arabidopsis. Transgenic Arabidopsis plants carrying En-1 elements were propagated for 12 generations using a single seed descent procedure. The distribution and activity of the En-1 element was monitored using Southern DNA hybridisations in generations 1, 6 and 12. In the first generation the highest number of En-1 insertions per line was 7, which increased to 20 in generation 12. The average number of En-1 insertions increased only slightly in the population, due to a gradual accumulation of segregants that lost the transposable element. During the development of the En-1 mutagenised population the element remained active even in the high-copy lines. In situ hybridisation demonstrated that multiple En-1 insertions were distributed over all Arabidopsis chromosomes. From the initial En-1 mutagenised populations many unstable gene mutations were recovered, indicating that En-1 can be used as a efficient tool for gene tagging in Arabidopsis.

Five ethylene-insensitive loci (wei1-wei5) were identified by using a low-dose screen for \"weak\" ethylene-insensitive mutants. wei1, wei2, and wei3 seedlings showed hormone insensitivity only in roots, whereas wei4 and wei5 displayed insensitivity in both roots and hypocotyls. The genes corresponding to wei1, wei4, and wei5 were isolated using a positional cloning approach. The wei1 mutant harbored a recessive mutation in TIR1, which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response, wei4, a dominant mutant, resulted from a mutation in the ethylene receptor ERS, whereas wei5, a semidominant mutant, was caused by a mutation in the EIN3-related transcription factor gene EIL1. The simultaneous loss of functional WEI5/EIL1 and EIN3 nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen Botrytis cinerea. Moreover, wei5/eil1 ein3 double mutants were able to fully suppress constitutive signaling caused by ctr1, suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants, wei2 and wei3 were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that wei2 and wei3 correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.

Polar auxin transport controls multiple developmental processes in plants, including the formation of vascular tissue. Mutations affecting the PIN-FORMED (PIN1) gene diminish polar auxin transport in Arabidopsis thaliana inflorescence axes. The AtPIN1 gene was found to encode a 67-kilodalton protein with similarity to bacterial and eukaryotic carrier proteins, and the AtPIN1 protein was detected at the basal end of auxin transport-competent cells in vascular tissue. AtPIN1 may act as a transmembrane component of the auxin efflux carrier.

The development of an Arabidopsis population via single-seed descent is described which includes 3,000 lines that carry approximately 15,000 independent insertions of the autonomous maize element En-1. A PCR strategy is outlined which allows the recovery of En-1-insertion mutants among this population in any random gene sequence of Arabidopsis thaliana. The method employs PCR reactions on pooled DNA. Positive amplification using a target-specific primer and an En-1-specific primer on row, column and single-tray pools identifies the putative insertion mutant. In a control experiment two insertion mutants of the PIN gene were successfully identified. In addition, a new independent insertion in the PIN gene was detected which was transmitted to the next generation and showed cosegregation with the pin phenotype. These examples demonstrate that the inheritance of inserts of the autonomous element En-1 is stable enough to make a proper genetic analysis feasible in a genomic background with multiple En-1 inserts.