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The Arabidopsis HYL1 gene encodes a nuclear double-stranded RNA-binding protein. A knockout mutation of the hyl1 gene is recessive and pleiotropic, causing developmental abnormalities, increasing sensitivity to abscisic acid, and reducing sensitivity to auxin and cytokinin. We report that levels of several microRNAs (miRNAs; miR159, -167, and -171) are reduced in homozygous mutant plants, and levels of two of three tested target mRNAs are elevated. Conversely, the miRNA levels are elevated in plants expressing a HYL1 cDNA from a strong promoter, and the corresponding target RNAs are reduced. These changes result from alterations in the stability of the target RNAs. However, doublestranded RNA-induced posttranscriptional gene silencing is unaffected by the hyl1 mutation. One-third to one-half of the cellular HYL1 protein is in a macromolecular complex, and a GFP-HYL1 fusion protein is found predominantly in the nucleus, although it is observed in both nucleus and cytoplasm in some cells. Within nuclei, HYL1 is associated with subnuclear bodies and ring-like structures. These observations provide evidence that the HYL1 protein is part of a nuclear macromolecular complex that is involved in miRNA-mediated gene regulation. Because hyl1 mutants show marked abnormalities in hormone responses, these results further suggest that miRNA-mediated changes in mRNA stability play a vital role in plant hormone signaling.

Arabidopsis thaliana plants with null mutations in the genes encoding the a and b subunits of the single heterotrimeric G protein are less and more sensitive, respectively, to O 3 damage than wild-type Columbia-0 plants. The first peak of the bimodal oxidative burst elicited by O 3 in wild-type plants is almost entirely missing in both mutants. The late peak is normal in plants lacking the Gb protein but missing in plants lacking the Ga protein. Endogenous reactive oxygen species (ROS) are first detectable in chloroplasts of leaf epidermal guard cells. ROS production in adjacent cells is triggered by extracellular ROS signals produced by guard cell membrane-associated NADPH oxidases encoded by the AtrbohD and AtrbohF genes. The late, tissue damage-associated component of the oxidative burst requires only the Ga protein and arises from multiple cellular sources. The early component of the oxidative burst, arising primarily from chloroplasts, requires signaling through the heterotrimer (or the Gbg complex) and is separable from Ga-mediated activation of membrane-bound NADPH oxidases necessary for both intercellular signaling and cell death.

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Proteins synthesized in the endoplasmic reticulum (ER) of eukaryotic cells must be folded correctly before translocation out of the ER. Disruption of protein folding results in the induction of genes for ER-resident chaperones, for example, BiP. This phenomenon is known as the ER stress response. We report here that bZIP60, an Arabidopsis thaliana basic leucine zipper (bZIP) transcription factor with a transmembrane domain, is involved in the ER stress response. When compared with wildtype Arabidopsis plants, homozygous bzip60 mutant plants show a markedly weaker induction of many ER stressresponsive genes. The bZIP60 protein resides in the ER membrane under unstressed condition and is cleaved in response to ER stress caused by either tunicamycin or DTT. The N-terminal fragment containing the bZIP domain is then translocated into the nucleus. Cleavage of bZIP60 is independent of the function of Arabidopsis homologs of mammalian S1P and S2P proteases, which mediate the proteolytic cleavage of the mammalian transcription factor ATF6. In Arabidopsis, expression of the bZIP60 gene and cleavage of the bZIP60 protein are observed in anthers in the absence of stress treatment, suggesting that the ER stress response functions in the normal development of active secretory cells.

Population growth, arable land and fresh water limits, and climate change have profound implications for the ability of agriculture to meet this century’s demands for food, feed, fiber, and fuel while reducing the environmental impact of their production. Success depends on the acceptance and use of contemporary molecular techniques, as well as the increasing development of farming systems that use saline water and integrate nutrient flows.

The results of genetic studies in Arabidopsis indicate that three proteins, the RNase III DICER-Like1 (DCL1), the dsRNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the C2H2 Zn-finger protein SERRATE (SE), are required for the accurate processing of microRNA (miRNA) precursors in the plant cell nucleus. To elucidate the biochemical mechanism of miRNA processing, we developed an in vitro miRNA processing assay using purified recombinant proteins. We find that DCL1 alone releases 21-nt short RNAs from dsRNA as well as synthetic miR167b precursor RNAs. However, correctly processed miRNAs constitute a minority of the cleavage products. We show that recombinant HYL1 and SE proteins accelerate the rate of DCL1-mediated cleavage of pre-and pri-miR167b substrates and promote accurate processing.microRNA ͉ biogenesis ͉ Dicer ͉ Arabidopsis M icro RNAs (miRNAs) are Ϸ21-nt regulatory RNAs found in viruses, plants, and animals. miRNAs inhibit gene expression by translational repression and by pairing with their target mRNA to promote their cleavage (1, 2). miRNA regulation is known to play an important role in development, stress responses, and carcinogenesis (3, 4). miRNAs are transcribed by RNA polymerase II as long primary transcripts, termed primiRNA, which are capped and polyadenylated (5, 6). In animals, the pri-miRNA, which contains the miRNA sequence embedded within a hairpin, is processed in the nucleus and the cytoplasm sequentially by two RNase III-family enzymes (7). The class II RNase III Drosha, together with the dsRNA-binding protein (dsRBP) DGCR8/Pasha, cleaves the stem loop of pri-miRNA in the nucleus to a hairpin RNA (pre-miRNA) of Ϸ70 nt (8-11). The pre-miRNAs are transported out of the nucleus by the Ran-binding protein exportin 5 (12-14). Dicer, another class III RNase III, cleaves the pre-miRNA in the cytoplasm to release the Ϸ22-nt miRNA/miRNA* duplex (15-17). Like Drosha, the animal Dicer has dsRBP partners, including Loquacious/ R3D1-L in Drosophila (18)(19)(20) and . There is also a Drosha-independent pathway for generating certain intronic miRNAs (24, 25).The plant miRNA biogenesis mechanism is somewhat different from that of animals. The pri-miRNAs transcripts appear to be RNA polII transcripts in plants, as they are in animals, but the hairpins are substantially more variable in length (26,27). Plants contain multiple Dicer homologs, termed the Dicer-like (DCL) enzymes. Of the four Dicer homologs in Arabidopsis, DCL1 carries out both cleavage steps in the nucleus to generate miRNAs (28-31). DCL2 produces 24-nt natural antisense siRNAs from complementary overlapping mRNA transcripts involved in the salt stress response (32). DCL3 generates the 24-nt DNA repeat sequence-associated siRNAs that direct heterochromatin formation (33), whereas DCL4 produces 21-nt transacting siRNAs that control leaf development (34-36). In addition, DCL4 and DCL2 generate 21-and 22-nt virus-derived small RNAs (vsRNA) redundantly functioning in antiviral defense (37).Animal Dicers and plant DCL1 enzymes are large prote...

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves ( hyl1 ), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N -(1-naphthyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1 , encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level. INTRODUCTIONThe development, growth, and survival of plants under a wide range of environmental conditions reflect an intricate interplay of physical and chemical conditions with the highly integrated sensing and response networks in plants. The growth habit and physiological properties of plants can differ markedly under different regimes of light, gravity, temperature, humidity, and salinity, among others. Hormones have long been known to be important internal mediating signals in plants, but the components of the underlying cellular machinery are just beginning to be identified and characterized (Trewavas and Malho, 1997; Grill and Himmelbach, 1998;Solano and Ecker, 1998;D'Agostino and Kieber, 1999). The range of proteins involved in receiving, transmitting, and responding to external signals includes receptorlike and other kinds of protein kinases, phosphatases, and transcription factors, as well as enzymes such as thioredoxin and farnesyltransferase, which influence protein structure or localization through mechanisms other than phosphorylation (Mulligan et al., 1997; Becraft, 1998;Bonetta and McCourt, 1998; Hooley, 1998;Bleecker, 1999;Thornton et al., 1999; Hirt, 2000;Urao et al., 2000).Ample physiological evidence supports the presence of interconnections among plant responses to different environmental stimuli; moreover, evidence is accumulating that certain mutations can simultaneously influence the response to more than one hormone or altered physical parameter (Wilson et al., 1990;Clouse et al., 1996;Nemeth et al., 1998;Ephritikhine et al., 1999a; Beaudoin et al., 2000; Ghassemian et al., 2000). The implication is that individual proteins can be responsible for such interconnections, either transmitting multiple signals or participating in distinct complexes that transmit different signals (Elio...