Seongbin Hwang

Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress

Summary Trehalose plays a role in drought stress resistance in a variety of organisms, including the extremely drought-tolerant «resurrection plants». Transgenic tobacco plants that produce trehalose were engineered by introduction of the Escherichia coli ots A and ots B genes, encoding trehalose-6-P synthase and trehalose-6-P phosphatase, respectively. The introduction of these genes had a pronounced effect on plant morphology and growth performance under drought stress. The transgenic Ots plants had larger leaves and altered stem growth. When grown under drought stress imposed by limiting water supply, the two transgenic tobacco lines Ots2 and Ots 5 yielded total dry weights that were 28 % and 39 % higher than those of wild-type tobacco. These increases in dry weight were due mainly to increased leaf production: leaf dry weights were up to 85 % higher for the best trehalose accumulator, Ots 5. No significant differences were observed under well-watered conditions. Chlorophyll fluorescence analysis of drought-stressed plants showed a higher photochemical quenching (qQ) and a higher ratio of variable fluorescence over maximal fluorescence (Fv/Fm), indicating a more efficient photosynthesis. The Ots 5 plants showed more negative leaf osmotic potentials than wild-type plants, particularly under drought stress, as well as higher levels of nonstructural carbohydrates; Ots2 plants showed intermediate values. Detached leaves from young, well-watered Ots plants had a better capacity than wild-type leaves to retain water when air-dried. They had lower osmotic potentials than wild-type leaves, and higher levels of glucose, fructose and sucrose.

CONTROL OF LHC GENE TRANSCRIPTION BY THE CIRCADIAN CLOCK IN CHLAMYDOMONAS REINHARDTII

Transcription of nuclear lhc genes has been shown to be under circadian clock control in angiosperms, but many aspects of this regulation have not been elucidated. Unicellular organisms, such as the green alga Chlamydomonas reinhardtii, offer significant advantages for the study of cellular clocks. Therefore, we have asked whether lhc gene expression is regulated by a circadian clock in C. reinhardtii. The mRNA for a photosystem I chlorophyll a/b apoprotein showed a strong diurnal rhythm in cells growing under 12 h/12 h light/dark (LD) cycles; the mRNA accumulated and then declined during the light period reaching very low levels at mid-dark. A similar diurnal pattern was documented for rbcS mRNA. In LD-grown cells shifted to continuous light, the ca. 24 h rhythm of lhca1 mRNA continued for at least 2 cycles. In LD-grown cells shifted to continuous darkness the rhythm of lhca1, but not rbcS2, mRNA also continued, although at lower absolute levels than in LD-grown cells. Also, in the cells shifted to continuous dark, the lhcal mRNA rhythm persisted in the absence of significant cell division. Pulselabelling with 32PO4 and sensitivity to actinomycin D demonstrated that control of lhca1 (and rbcS) is mainly transcriptional. However, it was also shown that the half-life of lhca1 mRNA (and rbcS2) is short (1–2h) and may also vary somewhat during a cycle. We conclude that a cellular, circadian clock regulates lhca1 transcription in C. reinhardtii.

Expression of yeast transcriptional activator MSN1 promotes accumulation of chromium and sulfur by enhancing sulfate transporter level in plants

MSN1 is a putative yeast transcriptional activator involved in chromium (Cr) accumulation. Here we show that overexpression of MSN1 enhances Cr and sulfur accumulation and Cr tolerance in transgenic tobacco. In addition, we found that expression of NtST1 (Nicotiana tabacum sulfate transporter 1) was elevated in MSN1‐ expressing transgenic tobacco, suggesting that chromate and sulfate are taken up via the sulfate transporter in plants. Supporting this, expression of NtST1 increased levels of Cr and S in Saccharomyces cerevisiae. Our findings suggest that yeast transcriptional activators can be used for developing effective metal remediators, and for improving the nutritional status of plants.

Requirement for cytoplasmic protein synthesis during circadian peaks of transcription of chloroplast-encoded genes in Chlamydomonas.

Cycloheximide, an inhibitor of cytoplasmic translation, induced a rapid reduction of 70–80% in levels of mRNA for the chloroplast elongation factor Tu (tufA) in asynchronously growing Chlamydomonas. This effect was shown to be mainly transcriptional, and not restricted to tufA, as transcription of other chloroplast-encoded genes were cycloheximide-sensitive, although not all equally (psbA showed no more than 40% inhibition). Confirmatory evidence that the inhibition of chloroplast transcription was mainly due to blocking cytoplasmic translation was obtained with the cycloheximide-resistant mutant act1, and by using another translation inhibitor, anisomycin. In synchronously growing Chlamydomonas, chloroplast transcription is regulated by the circadian clock, with the daily peak occurring during the early light period. When cycloheximide was added during this period, transcription was inhibited, but not when it was added during the trough period (late light to early dark). Moreover, in synchronized cells switched to continuous light, the drug blocked the scheduled increase in tufA mRNA, but did not remove the pre-existing mRNA. These experiments define two functionally different types of chloroplast transcription in Chlamydomonas, basal (cycloheximide-insensitive) and clock-induced (cycloheximide-sensitive), and indicate that the relative contribution of each type to the overall transcription of a given gene are not identical for all genes. The results also provide evidence for nuclear regulation of chloroplast transcription, thereby obviating the need for an organellar clock, at least for these rhythms.

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identifed in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [32P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.

Characterization of a cDNA encoding the 20-kDa photosystem I light-harvesting polypeptide of Chlamydomonas reinhardtii

A cDNA encoding the precursor to a major 20-kDa thylakoid polypeptide of Chlamydomonas reinhardtii (P22), previously localized to the photosystem I light-harvesting complex (LHCI), was characterized. N-terminal sequencing of P22 identified the precursor cleavage site. Genomic Southern blots and polymerase chain reaction analyses show that the gene for P22 (Lhca1*1) is single-copy and contains at least one intron. Northernblot analyses show that Lhca1*1 mRNA is highly regulated in light-dark synchronized cells. The primary sequence and predicted topology of P22 has features characteristic of light-harvesting chlorophyll a/b-binding proteins from higher plants. Sequence comparisons indicate that P22 has significantly greater identity with the Type-I LHCI protein of tomato, compared to other LHC proteins. This result suggests that the divergence of LHCI proteins into the classes found in higher plants may have occurred early in evolution, prior to the separation of green algae and land plants.

Expression of tobacco cDNA encoding phytochelatin synthase promotes tolerance to and accumulation of Cd and As inSaccharomyces cerevisiae

The first tobacco cDNA encoding phytochelatin synthase (NtPCS1) has been cloned by complementing the YCF1 (vacuolar ABC type transporter)-depleting yeast mutant DTY167 with an expression library fromNicotiana tabacum. When NtPCSI was over-expressed in DTY165 (WT) and DTY167 (mutant), tolerance to and the accumulation of cadmium (Cd) were enhanced. Interestingly, its expression promoted these responses as well to arsenic (As), but only in DTY167. We conclude thatNtPCS1 plays a role in tolerance to and the accumulation of both toxic metals inSaccharomyces cerevisiae.

The Density and Length of Root Hairs Are Enhanced in Response to Cadmium and Arsenic by Modulating Gene Expressions Involved in Fate Determination and Morphogenesis of Root Hairs in Arabidopsis

Root hairs are tubular outgrowths that originate from epidermal cells. Exposure of Arabidopsis to cadmium (Cd) and arsenic [arsenite, As(III)] increases root hair density and length. To examine the underlying mechanism, we measured the expression of genes involved in fate determination and morphogenesis of root hairs. Cd and As(III) downregulated TTG1 and GL2 (negative regulators of fate determination) and upregulated GEM (positive regulator), suggesting that root hair fate determination is stimulated by Cd and As(III). Cd and As(III) increased the transcript levels of genes involved in root hair initiation (RHD6 and AXR2) and root hair elongation (AUX1, AXR1, ETR1, and EIN2) except CTR1. DR5::GUS transgenic Arabidopsis showed a higher DR5 expression in the root tip, suggesting that Cd and As(III) increased the auxin content in the root tip. Knockdown of TTG1 in Arabidopsis resulted in increased root hair density and decreased root hair length compared with the control (Col-0) on 1/2 MS media. This phenotype may be attributed to the downregulation of GL2 and CTR1 and upregulation of RHD6. By contrast, gem mutant plants displayed a decrease in root hair density and length with reduced expression of RHD6, AXR2, AUX1, AXR1, ETR1, CTR1, and EIN2. Taken together, our results indicate that fate determination, initiation, and elongation of root hairs are stimulated in response to Cd and As(III) through the modulation of the expression of genes involved in these processes in Arabidopsis.

Tobacco phytochelatin synthase (NtPCS1) plays important roles in cadmium and arsenic tolerance and in early plant development in tobacco

Phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins, which are involved in heavy metal detoxification in plants and other living organisms. Previously, we cloned a PCS1 gene from tobacco (Nicotiana tabacum) and showed that its expression in yeast (Saccharomyces cerevisiae) resulted in increased cadmium (Cd) tolerance and Cd accumulation (Kim et al., J Plant Biol 48:440–447, 2005). To examine the role of NtPCS1 in tobacco, we generated transgenic tobacco lines over-expressing NtPCS1 in the sense or antisense direction. Compared with other PCS1-expressing plants, NtPCS1-expressing tobacco exhibited a unique phenotype: increased tolerance to cadmium and arsenite, but no change in cadmium and arsenic accumulation. In addition, the antisense-NtPCS1 tobacco lines showed growth retardation in the early stage, suggesting that phytochelatin also plays a role in plant development. These results demonstrate that NtPCS1 plays important roles in metal(loid) tolerance as well as in growth and development in tobacco.

Overexpression of NtUBQ2 encoding Ub-extension protein enhances cadmium tolerance by activating 20S and 26S proteasome in tobacco (Nicotiana tabacum)

The Ub/26S proteasome system removes abnormal proteins and most short-lived regulatory proteins, thereby contributing to cell proliferation, hormone responses, development and resistance to abiotic and biotic stresses. Here we show that cadmium tolerance is related positively to the 20S proteasome (catalytic particle of 26S proteasome) activity in plants. By transforming WT yeast Y800 with a tobacco expression cDNA library, we isolated a tobacco cDNAs, NtUBQ2 (the Ub-extension protein) conferring cadmium tolerance. Overexpression of NtUBQ2 increased cadmium tolerance in transgenic tobacco; 20S proteasome activity was enhanced and ubiquitinated protein level was diminished in response to cadmium. In contrast, proteasome activity was reduced and ubiquitinated protein level was less decreased than transgenic tobacco by Cd treatment in control tobacco which is sensitive to Cd. These observations strongly suggest that plants acquire cadmium tolerance by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or Ub-independent 20S proteasome. This finding could be applied to engineering efficient metal phytoremediators.