A number of observations have implicated auxin in the formation of vascular tissues in plant organs. These include vascular strand formation in response to local auxin application, the effects of impaired auxin transport on vascular patterns and suggestive phenotypes of Arabidopsis auxin response mutants. In this study, we have used molecular markers to visualize auxin response patterns in developing Arabidopsis leaves as well as Arabidopsis mutants and transgenic plants to trace pathways of auxin signal transduction controlling the expression of early procambial genes. We show that in young Arabidopsis leaf primordia, molecular auxin response patterns presage sites of procambial differentiation. This is the case not only in normal development but also upon experimental manipulation of auxin transport suggesting that local auxin signals are instrumental in patterning Arabidopsis leaf vasculature. We further found that the activity of the Arabidopsis gene MONOPTEROS, which is required for proper vascular differentiation, is also essential in a spectrum of auxin responses, which include the regulation of rapidly auxin-inducible AUX/IAA genes, and discovered the tissue-specific vascular expression profile of the class I homeodomain-leucine zipper gene, AtHB20. Interestingly, MONOPTEROSactivity is a limiting factor in the expression of AtHB8and AtHB20, two genes encoding transcriptional regulators expressed early in procambial development. Our observations connect general auxin signaling with early controls of vascular differentiation and suggest molecular mechanisms for auxin signaling in patterned cell differentiation.

Homeodomain‐leucine zipper (HD‐Zip) proteins are putative transcription factors encoded by a class of recently discovered homeobox genes as yet found only in plants. This paper reports on the characterization of one of these genes, ATHB‐7, in Arabidopsis thaliana. ATHB‐7 transcripts were present in all organs of the plant at low levels, but expression was induced several‐fold by water deficit, osmotic stress as well as by exogenous treatment with abscisic acid (ABA), a response being detectable at 10⁻⁸ M and reaching a maximum at 10⁻⁶ M ABA. The ATHB‐7 transcript was detected within 30 min after treatment with ABA and the transcript level was rapidly reduced after removal of the hormone. The induction of ATHB‐7 was shown to be mediated strictly via ABA, since no induction of ATHB‐7 was detectable in the ABA‐deficient mutant aba‐3 subjected to drought treatment. Induction levels in two ABA‐insensitive mutants abi2 and abi3 were similar to the wild‐type response. In the abi1 mutant, however, induction was impaired as 100‐fold higher concentrations of ABA were required for a maximum induction as compared with wild‐type. In this mutant the ATHB‐7 response was reduced also after drought and osmotic stress treatments. These results indicate that ATHB‐7 is transcriptionally regulated in an ABA‐dependent manner and may act in a signal transduction pathway which mediates a drought response and also includes ABI1.

Summary: Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN‐FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non‐polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization‐of‐auxin‐flow hypothesis.

Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants approximately 260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes.