Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for β-amylase and on the accumulation of anthocyanin that are inducible by sugars

We identified a mutant of Arabidopsis thaliana ecotype Col-0 in which significantly reduced levels of expression of the gene for β-amylase ( ATβ-Amy ) were detected in leaves in response to high concentrations of sucrose, glucose or fructose. Genetic studies, including a cross with transgenic plants that harbored the ATβ-Amy:GUS transgene with the promoter of ATβ-Amy , indicated that this phenotype was caused by a recessive mutation, Iba1 , that affected expression of ATβ-Amy in trans . We also found a reduced level of sugar-induced expression of ATβ-Amy in the Landsberg erecta (L er ) ecotype compared with other ecotypes. This phenotype seemed to be due to a recessive trait, provisionally designated Iba2 , that was linked to neither erecta nor Iba1 . The Iba2 mutation also affected expression of ATβ-Amy:GUS transgene. Accumulation of starch and sugars after treatment of leaves with sucrose was not affected in the Iba1 mutant and L er plants. However, both Iba1 mutant and L er plants accumulated low levels of anthocyanin in response to sucrose, results that suggested the existence of some genetic linkage between regulation of the expression of ATβ-Amy and regulation of the accumulation of anthocyanin. Although the Iba1 and Iba2 mutations did not affect sugar-inducible gene expression in general, the expression of sugar-regulated genes other than the gene for β-amylase was differentially affected in the Iba1 mutant and L er plants. These results suggest that the sugar-regulated expression of many genes in plants might be mediated by multiple signal-transduction pathways.     

Shade avoidance and the regulation of leaf inclination in Arabidopsis

As a rosette plant, Arabidopsis thaliana forms leaves near to the ground, which causes the plant to be vulnerable to shading by neighbours. One mechanism to avoid such shading is the regulation of leaf inclination, such that leaves can be raised to more vertical orientations to prevent neighbouring leaves from overtopping them. Throughout Arabidopsis rosette development, rosette leaves move to more vertical orientations when shaded by neighbouring leaves, exposed to low light levels or placed in the dark. After dark-induced reorientation of leaves, returning them to white light causes the leaves to reorient to more horizontal inclinations. These light-dependent leaf movements are more robust than, and distinct from, the diurnal movements of rosette leaves. However, the movements are gated by the circadian clock. The light-dependent leaf orientation response is mediated primarily through phytochromes A, B and E, with the orientation varying with the ratio of red light to far-red light, consistent with other shade-avoidance responses. However, even plants lacking these phytochromes were able to alter leaf inclination in response to white light, suggesting a role for other photoreceptors. In particular, we found significant changes in leaf inclination for plants exposed to green light. This green light response may be caused, in part, by light-dependent regulation of abscisic acid (ABA) biosynthesis.     

The Arabidopsis homeobox gene, ATHB16, regulates leaf development and the sensitivity to photoperiod in Arabidopsis

This report describes the characterisation of ATHB16, a novel Arabidopsis thaliana homeobox gene, which encodes a homeodomain-leucine zipper class I (HDZip I) protein. We demonstrate that ATHB16 functions as a growth regulator, potentially as a component in the light-sensing mechanism of the plant. Endogenous ATHB16 mRNA was detected in all organs of Arabidopsis, at highest abundance in rosette leaves. Reduced levels of ATHB16 expression in transgenic Arabidopsis plants caused an increase in leaf cell expansion and consequently an increased size of the leaves, whereas leaf shape was unaffected. Transgenic plants with increased ATHB16 mRNA levels developed leaves that were smaller than wild-type leaves. Therefore, we suggest ATHB16 to act as a negative regulator of leaf cell expansion. Furthermore, the flowering time response to photoperiod was increased in plants with reduced ATHB16 levels but reduced in plants with elevated ATHB16 levels, indicating that ATHB16 has an additional role as a suppressor of the flowering time sensitivity to photoperiod in wild-type Arabidopsis. As deduced from the response of transgenic plants with altered levels of ATHB16 expression in hypocotyl elongation assays, the gene may act to regulate plant development as a mediator of a blue light response.     

A novel ER-derived compartment,the ER body,selectively accumulates a beta-glucosidase with an ER-retention signal in Arabidopsis

The ER body is a novel compartment that is derived from endoplasmic reticulum (ER) in Arabidopsis. In contrast to whole seedlings which have a wide distribution of the ER bodies, rosette leaves have no ER bodies. Recently, we reported that wound stress induces the formation of many ER bodies in rosette leaves, suggesting that the ER body plays a role in the defense system of plants. ER bodies were visualized in transgenic plants (GFP-h) expressing green fluorescent protein (GFP) with an ER-retention signal, HDEL. These were concentrated in a 1000-g pellet (P1) of GFP-h plants. We isolated an Arabidopsis mutant, nai1, in which fluorescent ER bodies were hardly detected in whole plants. We found that a 65-kDa protein was specifically accumulated in the P1 fraction of GFP-h plants, but not in the P1 fraction of nai1 plants. N-terminal peptide sequencing revealed that the 65-kDa protein was a beta-glucosidase, PYK10, with an ER-retention signal, KDEL. Immunocytochemistry showed that PYK10 was localized in the ER bodies. Compared with the accumulation of GFP-HDEL, which was associated with both cisternal ER and ER bodies, the accumulation of PYK10 was much more specific to ER bodies. PYK10 was one of the major proteins in cotyledons, hypocotyls and roots of Arabidopsis seedlings, while PYK10 was not detected in rosette leaves that have no ER bodies. These findings indicated that PYK10 is the main component of ER bodies. It is possible that PYK10 produces defense compounds when plants are damaged by insects or wounding.     

Response to darkness of late-responsive dark-inducible genes is positively regulated by leaf age and negatively regulated by calmodulin-antagonist-sensitive signalling in Arabidopsis thaliana

Induction after prolonged darkness distinguishes the late-responsive genes din2 and din9 from the early-responsive gene din3 in Arabidopsis. The former genes were coincidently induced with the senescence marker gene YLS4 in rosette leaves of different ages and in the early-senescence mutant hys1. The calmodulin antagonists W-7, trifluoperazine, and fluphenazine accelerated the expression of the former genes in darkness but not in light, and had little effect on the latter gene. Our results suggest that Ca(2+)/calmodulin signalling conveys a negative signal that suppresses the responses of late-responsive din genes to prolonged darkness. The results are discussed in relation to natural senescence.     

Phytochrome A, phytochrome B and other phytochrome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants

The expression of the Arabidopsis ATHB-2 gene is light-regulated both in seedlings and in adult plants. The gene is expressed at high levels in rapidly elongating etiolated seedlings and is down-regulated by a pulse of red light (R) through the action of a phytochrome other than phytochrome A or B, or by a pulse of far-red light (FR) through the action of phytochrome A. In green plants, the expression of the ATHB-2 gene is rapidly and strongly enhanced by lowering the R:FR ratio perceived by a phytochrome other than A or B. Returning the plant to a high R:FR ratio results in an equally rapid decrease of the ATHB-2 mRNA. Consistently, plants overproducing ATHB-2 show developmental phenotypes characteristic of plants grown in low R:FR: elongated petioles, reduced leaf area, early flowering, and reduced number of rosette leaves. Taken together, the data strongly suggest a direct involvement of ATHB-2 in light-regulated growth phenomena throughout Arabidopsis development.     

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.     

Wounding changes the spatial expression pattern of the arabidopsis plastid omega-3 fatty acid desaturase gene (FAD7) through different signal transduction pathways.

The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids in membrane lipids. The mRNA levels of the Arabidopsis FAD7 gene in rosette leaves rose rapidly after local wounding treatments. Wounding also induced the expression of the FAD7 gene in roots. To study wound-responsive expression of the FAD7 gene in further detail, we analyzed transgenic tobacco plants carrying the -825 Arabidopsis FAD7 promoter-beta-glucuronidase fusion gene. In unwounded transformants, FAD7 promoter activity was restricted to the tissues whose cells contained chloroplasts. Activation of the FAD7 promoter by local wounding treatments was more substantial in stems (29-fold) and roots (10-fold) of transgenic plants than it was in leaves (approximately two-fold). Significant induction by wounding was observed in the overall tissues of stems and included trichomes, the epidermis, cortex, vascular system, and the pith of the parenchyma. Strong promoter activity was found preferentially in the vascular tissues of wounded roots. These results indicate that wounding changes the spatial expression pattern of the FAD7 gene. Inhibitors of the octadecanoid pathway, salicylic acid and n-propyl gallate, strongly suppressed the wound activation of the FAD7 promoter in roots but not in leaves or stems. In unwounded plants, exogenously applied methyl jasmonate activated the FAD7 promoter in roots, whereas it repressed FAD7 promoter activity in leaves. Taken together, wound-responsive expression of the FAD7 gene in roots is thought to be mediated via the octadecanoid pathway, whereas in leaves, jasmonate-independent wound signals may induce the activation of the FAD7 gene. These observations indicate that wound-responsive expression of the FAD7 gene in aerial and subterranean parts of plants is brought about by way of different signal transduction pathways.     

Negative regulation in the expression of a sugar-inducible gene in Arabidopsis thaliana. A recessive mutation causing enhanced expression of a gene for beta-amylase.

Expression of a beta-amylase gene of Arabidopsis thaliana (AT beta-Amy) is regulated by sugars. We identified a mutant, hba1, in which the level of expression of AT beta-Amy in leaves of plants that had been grown in a medium with 2% sucrose was significantly higher than that in wild-type plants. Higher that wild-type levels of beta-amylase in hba1 plants depended on the presence of 1 to 2% sucrose or 1% glucose in the medium, whereas leaves of mutant plants grown with higher levels of sugars had beta-amylase activities similar to those in leaves of wild-type plants. The hba1 phenotype was recessive and did not affect levels of sugars and starch in leaves. It is proposed that expression of AT beta-Amy is regulated by a combination of both positive and negative factors, dependent on the level of sugars, and that HBA1 might function to maintain low-level expression of AT beta-Amy until the level of sugars reaches some high level. Results of crosses of hba1 plants with transgenic plants that harbored an AT beta-Amy:GUS transgene with 1587 bp of the 5'-upstream region suggested that HBA1 affects expressions of AT beta-Amy in trans. The hba1 plants also had growth defects and elevated levels of anthocyanin in their petioles. However, sugar-related changes in levels of several mRNAs other than beta-amylase mRNA were unaffected in hba1 plants, suggesting that only a subset of sugar-regulated genes is under the control HBA1.     

Characterization of an ultraviolet B-induced lipase in Arabidopsis

An Arabidopsis expressed sequence tag clone, 221D24, encoding a lipase has been characterized using an antisense approach. The lipase gene is expressed during normal growth and development of Arabidopsis rosette leaves but is down-regulated as the leaves senesce. When plants are exposed to sublethal levels of UV-B radiation, expression of the lipase is strongly up-regulated. The lipase protein is localized in the cell cytosol and is present in all organs of Arabidopsis plants. Recombinant lipase protein produced in Escherichia coli preferentially hydrolyzed phospholipids, indicating that the gene encodes a phospholipase. Transgenic plants in which lipase expression is suppressed showed enhanced tolerance to UV-B stress but not osmotic stress and were unable to up-regulate PR-1 expression when irradiated with UV-B. The observations collectively indicate that the lipase is capable of deesterifying membrane phospholipids and is up-regulated in response to UV-B irradiation.     

The ER body,a novel endoplasmic reticulum-derived structure in Arabidopsis

Plant cells develop various endoplasmic reticulum (ER)-derived structures with specific functions. The ER body, a novel ER-derived compartment in Arabidopsis, is a spindle-shaped structure (approximately 10 microm long and approximately 1 microm wide) that is surrounded by ribosomes. Similar structures were found in many Brassicaceae plants in the 1960s and 1970s, but their main components and biological functions have remained unknown. ER bodies can be visualized in transgenic Arabidopsis expressing the green fluorescent protein with an ER-retention signal. A large number of ER bodies are observed in cotyledons, hypocotyls and roots of seedlings, but very few are observed in rosette leaves. Recently nai1, a mutant that does not develop ER bodies in whole seedlings, was isolated. Analysis of the nai1 mutant reveals that a beta-glucosidase, called PYK10, is the main component of ER bodies. The putative biological function of PYK10 and the inducibility of ER bodies in rosette leaves by wound stress suggest that the ER body functions in the defense against herbivores.     

Characterization of mutants in Arabidopsis showing increased sugar-specific gene expression, growth, and developmental responses

Sugars such as sucrose serve dual functions as transported carbohydrates in vascular plants and as signal molecules that regulate gene expression and plant development. Sugar-mediated signals indicate carbohydrate availability and regulate metabolism by co-coordinating sugar production and mobilization with sugar usage and storage. Analysis of mutants with altered responses to sucrose and glucose has shown that signaling pathways mediated by sugars and abscisic acid interact to regulate seedling development and gene expression. Using a novel screen for sugar-response mutants based on the activity of a luciferase reporter gene under the control of the sugar-inducible promoter of the ApL3 gene, we have isolated high sugar-response (hsr) mutants that exhibit elevated luciferase activity and ApL3 expression in response to low sugar concentrations. Our characterization of these hsr mutants suggests that they affect the regulation of sugar-induced and sugar-repressed processes controlling gene expression, growth, and development in Arabidopsis. In contrast to some other sugar-response mutants, they do not exhibit altered responses to ethylene or abscisic acid, suggesting that the hsr mutants may have a specifically increased sensitivity to sugars. Further characterization of the hsr mutants will lead to greater understanding of regulatory pathways involved in metabolite signaling.     

Leaf-specific suppression of deoxyhypusine synthase in Arabidopsis thaliana enhances growth without negative pleiotropic effects

Deoxyhypusine synthase (DHS) mediates the first of two enzymatic reactions required for the post-translational activation of eukaryotic translation initiation factor 5A (eIF5A), which in turn is thought to facilitate translation of specific mRNAs. Analyses of GUS activity in transgenic Arabidopsis plants expressing the GUS reporter gene under regulation of the promoter for AtDHS revealed that the expression of DHS changes both spatially and temporally as development progresses. In particular, DHS is expressed not only in rosette leaves, but also in the anthers of developing flowers. To determine the role of DHS in leaves, transgenic plants in which DHS was selectively suppressed in rosettes of Arabidopsis plants were prepared. This was achieved by expressing AtDHS 3'-UTR cDNA as a transgene under regulation of the promoter for AtRbcS2, a gene encoding the small subunit of Rubisco. The dominant phenotypic traits of the DHS-suppressed plants proved to be a dramatic enhancement of both vegetative and reproductive growth. As well, the onset of leaf senescence in the DHS-suppressed plants was delayed by approximately 1 week, but there was no change in the timing of bolting. In addition, there was no evidence for the negative pleiotropic effects, including stunted reproductive growth and reduced seed yield, noted previously for transgenic plants in which DHS was constitutively suppressed. The results indicate that DHS plays a pivotal role in both growth and senescence.