Sugar Coordinately and Differentially Regulates Growth- and

Sugar Coordinately and Differentially RegulatesGrowth- and Stress-Related Gene Expression via aComplex Signal Transduction Network and MultipleControl Mechanisms1

Shin-Lon Ho, Yu-Chan Chao, Wu-Fu Tong, and Su-May Yu*

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China (S.-L.H.,Y.-C.C., S.-M.Y.); and Department of Biology, National Taiwan Normal University, Taipei, Taiwan 10764,Republic of China (S.-L.H., W.-F.T.)

In plants, sugars are required to sustain growth and regulate gene expression. A large set of genes are either up- ordown-regulated by sugars; however, whether there is a common mechanism and signal transduction pathway for differ-ential and coordinated sugar regulation remain unclear. In the present study, the rice (Oryza sativa cv Tainan 5) cell culturewas used as a model system to address this question. Sucrose and glucose both played dual functions in gene regulation asexemplified by the up-regulation of growth-related genes and down-regulation of stress-related genes. Sugar coordinatelybut differentially activated or repressed gene expression, and nuclear run-on transcription and mRNA half-life analysesrevealed regulation of both the transcription rate and mRNA stability. Although coordinately regulated by sugars, thesegrowth- and stress-related genes were up-regulated or down-regulated through hexokinase-dependent and/or hexokinase-independent pathways. We also found that the sugar signal transduction pathway may overlap the glycolytic pathway forgene repression. a-Amylase and the stress-related genes identified in this study were coordinately expressed under sugarstarvation, suggesting a convergence of the nutritional and environmental stress signal transduction pathways. Together,our studies provide a new insight into the complex signal transduction network and mechanisms of sugar regulation ofgrowth and stress-related genes in plants.

In plants, sugars do not only function as metabolicresources and structural constituents of cells, theyalso act as important regulators of various processesassociated with plant growth and development. Avariety of genes, whose products are involved indiverse metabolic pathways and cellular functions,are either induced or repressed depending upon theavailability of soluble sugars. In general, sugars favorthe expression of enzymes in connection with biosyn-thesis, use, and storage of reserves (including starch,lipid, and proteins), while repressing the expressionof enzymes involved in photosynthesis and reservemobilization (Koch, 1996). A large and specific set ofgenes has been reported to be positively regulated bysugars. Examples include: (a) genes that encode stor-age proteins, e.g. patatin in potato and sporamin insweet potato (Hattori et al., 1990; Jefferson et al.,1990); (b) genes that encode proteins related to starchbiosynthesis, e.g. ADP-Glc pyrophosphorylase (Muller-Rober et al., 1990); (c) genes that encode defenseproteins, e.g. proteinase inhibitor II in potato (Kim etal., 1991); and (d) genes that encode proteins for Sucmetabolism, e.g. invertase and Suc synthase (Sus1)

(Koch et al., 1992; Roitsch et al., 1995). In contrast, avariety of genes are negatively regulated by sugars,and their expression is induced by sugar deprivation,e.g. sugar represses expression of a-amylase genes inrice (Oryza sativa cv Tainan 5) suspension cells andgerminating embryos (Yu et al., 1991, 1996); endo-peptidase, Suc synthase (Sh1), and Asn synthasegenes in maize root tips (Koch et al., 1992; James etal., 1993; Chevalier et al., 1995); and malate synthaseand isocitrate lyase genes in cucumber cotyledon andsuspension cells (Graham et al., 1994). It is not knownwhether a common mechanism is responsible fordifferential sugar regulation.

Plants are considered to be carbon autotrophs, butthey can be considered as carbon heterotrophs dur-ing some part of their life cycle or in some of theirnon-green organs like roots, stems, and flowers thatare not involved in photosynthesis. Furthermore, car-bohydrate depletion can occur and is a fact of life inmost plants. For instance, variations in environmen-tal factors, such as light, water, or temperature, andattacks by pathogens or herbivores may lead to asignificant decrease in the efficiency of photosynthe-sis in source tissues and thus reduce the supply ofcarbohydrates to sink tissues. Under conditions ofsugar deprivation, substantial physiological and bio-chemical changes occur to sustain respiration andother metabolic processes (Yu, 1999a). When Suc isomitted from the nutrient medium of cell cultures or

1 This work was supported by the Academia Sinica, the NationalScience Council (grant no. NSC 89 –2311–B– 001– 023), and theBiomedical Research Foundation of the Republic of China.

* Corresponding author; e-mail [email protected]; fax886 –2–2788 –2695 or 886 –2–2782– 6085.

Plant Physiology, February 2001, Vol. 125, pp. 877–890, www.plantphysiol.org © 2001 American Society of Plant Physiologists 877

Dow

nloaded from https://academ

ic.oup.com/plphys/article/125/2/877/6099771 by guest on 14 N

ovember 2021

isolated tissues, cell growth ceases and the cellularstarch and sugar levels, respiration rates, and meta-bolic activities dramatically decline (Journet et al.,1986; Brouquisse et al., 1992; Chen et al., 1994). Sugarstarvation may also trigger an autophagic processinvolved in the regression of cytoplasm, includingorganelles and in the recycling of respiratory sub-strates (Chen et al., 1994; Aubert et al., 1996; Yu,1999a).

Understanding the mechanisms involved in sugarsignal transduction and sugar regulation of gene ex-pression in plants is still in its early stages. Moststudies on the mechanisms of sugar activation andsugar repression in plants have emphasized regula-tion at the transcriptional level (Sheen, 1990; Chan etal., 1994; Graham et al., 1994; Lu et al., 1998). How-ever, sugar repression of a-amylase gene expressioninvolves control of both transcription and mRNAstability (Sheu et al., 1994, 1996). A sugar responsecomplex (SRC) in the promoter region of a Suc de-privation highly inducible rice a-amylase gene,aAmy3, has been identified. This SRC contains threeessential motifs for a high level of sugar starvation-induced gene expression in rice cells (Lu et al., 1998).Studies on aAmy3 mRNA have also identified essen-tial sequences in its 39-untranslated region (39-UTR)that control sugar-dependent mRNA stability (Chanand Yu, 1998a, 1998b). The 39-UTR of a cell wallinvertase gene (Incw1) recently was shown to be in-volved in translational control of Incw1 by sugars incultured maize suspension cells (Cheng et al., 1999).

A considerable amount of information concerningthe sugar signal transduction pathway is availablefrom research in yeast (Carlson, 1987; Entian andBarnett, 1992; Gancedo, 1992). However, very fewyeast homologs in plants have been studied or shownto serve similar functions or to be regulated in amanner similar to the yeast system. Hexokinase, theenzyme that catalyzes the phosphorylation of hexosesugars at the first step of the glycolytic pathway, hasbeen implicated as a Glc sensor in organisms asdiverse as yeast (Rose et al., 1991) and mammals(Efrat et al., 1994). Recent studies suggest that hex-okinase also acts as the primary sugar sensor inplants (Jang et al., 1997; Smeekens and Rook, 1997).However, multiple sugar sensing pathways have alsobeen proposed to exist in plants (Halford et al., 1999;Sheen et al., 1999; Smeekens, 2000). A gene (SnRK1)encoding the yeast Ser/Thr protein kinase (SNF1)homolog isolated from potato was recently shown tobe required for activation of Suc synthase gene ex-pression (Purcell et al., 1998). Whether SnRK1 activ-ity is regulated by Glc or some other hexose andwhether SnRK1 plays a role in the derepression ofsugar-repressible genes in plants as in yeast (Ronne,1995) are not known.

Expression of genes regulated by sugars can also beaffected by various other factors, such as light(Sheen, 1990), phosphate (Sadka et al., 1994), hor-

mones (DeWald et al., 1994; Zhou et al., 1998), patho-gen infection (Herbers et al., 1996), as well as wound-ing and anaerobiosis (Salanoubat and Belliard, 1989).The mechanisms underlying the crosstalk betweensugar and other signal transduction pathways andgene regulation are not clear. It will be interesting todetermine whether different internal and externalsignals are integrated to result in the coordinatedregulation of gene expression. The aim of the presentstudy was to explore whether there is a commonmechanism for differential and coordinated sugarregulation and to better understand the mechanismthat connects the sensing and transmission of sugarsignals with the regulation of gene expression inplants. Rice suspension cell culture, which is readilyamenable to exogenous metabolic manipulations,was used as a model system for these studies. Theusefulness of this type of cell culture in such studiesis well documented (Graham et al., 1994; Ehness etal., 1997; Cheng et al., 1999; Yu, 1999a). In the presentstudies, some growth- and stress-related genes up-and down-regulated by Suc, respectively, were iden-tified and found to be differentially and coordinatelyregulated by Suc, and the regulation involves controlof both transcription rate and mRNA stability.Hexokinase-dependent and -independent pathwayswere found to be involved in up- and down-regulation of gene expression. Based on several linesof experimental evidence, we propose two potentialcomplex signal transduction networks for differentialand coordinated regulation of gene expression: onewhich connects the sugar signal transduction path-way to the sugar metabolic pathway and one whichconnects the sugar starvation signal to the stresssignals.

RESULTS

Sugars Have Dual Functions in Gene Regulation

To study the mechanism that switches on and offsugar-dependent gene expression, it was necessary toidentify genes whose expression are up- or down-regulated by sugars. Cells were first provided withSuc for 72 h, starved of Suc for 72 h, then providedwith Suc for 24 h. Total RNA was purified from thesecells and subjected to gel-blot analysis. SeveralcDNAs that encode proteins known to be requiredfor cell growth, e.g. actin (Act), glyceraldehyde-3-phosphate dehydrogenase (G3PD), Histone H3 (His),and Suc synthase P-2 (SSP2), as well as various pro-teins related to stress response, e.g. alcohol dehydro-genase (ADH2), heat shock protein 86 (HSP86), andubiquitin precursor (Ubi), were used as probes forthe gel-blot analysis. The mRNA levels of a-amylasegenes (a-Amy) increased after the onset of Suc star-vation and decreased as cells were provided with Suc(Fig. 1a). These observations were consistent with ourprevious report (Sheu et al., 1994). In contrast, themRNA levels of Act, ADH2, G3PD, His, HSP86, and

Ho et al.

878 Plant Physiol. Vol. 125, 2001

Dow



ovember 2021

SSP2 genes were initially high in Suc-provided cellsand then decreased significantly after Suc starvation.Somewhat different expression patterns were ob-served for His and HSP86 genes. Accumulation of theHis mRNA was dramatically and transiently in-creased 1 h after shifting cells from Suc-free to Suc-containing medium (Fig. 1a, lane 15). Accumulationof HSP86 mRNA progressively decreased andreached the lowest level 8 h after starvation (Fig. 1a,lane 9) and progressively increased afterward whilecells remained under starvation. Three hybridizationsignals were observed for Ubi genes: one decreasedafter Suc starvation, one increased after Suc starva-tion, and the other one increased with culture age.

To identify genes whose expression is down-regulated by sugars, we performed a differentialscreening of a cDNA library constructed from poly(A1)mRNA prepared from 4-h Suc-starved rice cells. Morethan 100 cDNA clones whose expression increased afterSuc starvation were isolated. Twelve cDNA cloneswhose signals were significantly stronger by hybrid-ization with cDNA probes of Suc-starved cells com-pared with cDNA probes of Suc-provided cells wereselected for further characterization. By hybridiza-tion with the aAmy8-C probe, three of the cDNAswere found to be a-amylase genes and were notfurther characterized. Partial 39-end nucleotide se-quence analyses of the other nine cDNAs suggestedthat they were derived from six different genes. Toconfirm preferential induction by sugar starvation,the six selected genes were used as probes to hybrid-ize to replicated gel blots of total cellular RNA asshown in Figure 1a. mRNA levels of these genes werelow or almost undetectable in Suc-provided cells andincreased in Suc-starved cells (Fig. 1b). Two hybrid-ization signals were observed for clone E1: After Sucstarvation, one increased only at early stages, but theother one increased progressively with time. Nucle-otide sequences of these sugar down-regulatedcDNAs were compared with sequence data in Gen-Bank. cDNA B1 is highly homologous to a Gly-richRNA-binding protein gene from rice (Macknight etal., 1998). cDNAs A2, D1, E1, and F1 are not identicalbut are similar and highly homologous to a geneencoding the virus-inducible Gly-rich cell wall proteinfrom rice (Fang et al., 1991; Lei and Wu, 1991). cDNAA3 is highly homologous to a salt and drought stress-inducible gene, salT, from rice (Claes et al., 1990).

To determine whether accumulation of proteins ofthe sugar-regulated genes parallel mRNA levels, pro-tein gel-blot analyses were performed using threeantibodies against a-Amy, Act, and SSP2, respec-tively. As shown in Figure 2, the levels of a-amylasesincreased in Suc-starved cells and decreased in Suc-

Figure 1. Two groups of genes are coordinately regulated but in anopposite manner by sugars. a, RNA gel-blot analysis of growth-related genes. Rice suspension cells were cultured in Suc-containing(1S) medium for 72 h, transferred to Suc-free (2S) medium for 72 h,and transferred to 1S medium for an additional 24 h. Total RNA waspurified and fractionated on two duplicated 1% (w/v) agarose gels.Five membrane blots were parallel prepared and sequentially hybrid-ized with indicated probes. The Act probe, His and Ubi probes,G3PD probe, HSP86 and ADH2 probes, and a-Amy and SSP2 probeswere respectively hybridized to each of the five blots. The a-Amy andAct cDNA probes hybridized to the mRNAs of all a-amylase and

actin genes, respectively (Sheu et al., 1996). b, RNA gel-blot analysisof stress-related genes. Five membrane blots were parallel prepared.The A2 probe, B1 probe, A3 probe, D1 and F1 probes, and E1 probewere respectively hybridized to each of the five blots.

Sugar Regulation of Growth- and Stress-Related Genes

Plant Physiol. Vol. 125, 2001 879

Dow



ovember 2021

provided cells. In contrast, the levels of Act and SSP2decreased in Suc-starved cells and increased in Suc-provided cells.

Sugars Alter Gene Transcription

To define the mechanism of sugar-dependent up-and down-regulation of gene expression, the tran-scription rates of individual genes were determined.Nuclear run-on transcription analyses were per-formed with nuclei isolated from cells provided withor without Suc for 24 h. The transcription rates ofrRNA genes in Suc-provided and Suc-starved cells(Fig. 3a, slot 9 and Fig. 3b, slot 10) were kept at thesame levels and the transcription rates of the remain-ing genes were compared. For sugar up-regulated(SU) genes, only the transcription rates of G3PD andADH2 genes were higher in Suc-provided cells thanin Suc-starved cells (Fig. 3a, slots 4 and 5). It issurprising that the transcription rates of His, HSP86,and Act genes were lower in Suc-provided cells thanin Suc-starved cells (Fig. 3a, slots 1, 2, and 7). Theresult of Act was consistent with our previous report(Sheu et al., 1994). The transcription rate of the SSP2gene remained unchanged (Fig. 3a, slot 3). On theother hand, for all the sugar down-regulated (SD)genes, the transcription rates were higher in Suc-starved cells than in Suc-provided cells (Fig. 3b). Thetranscription rate of a-Amy was significantly higherin Suc-starved cells than in Suc-provided cells (Fig.3a, slot 6 and Fig. 3b, slot 7), which was consistentwith our previous study (Sheu et al., 1994). Theseresults demonstrate that sugar down-regulation ofgene expression involves transcriptional control,whereas sugar up-regulation of gene expression mayor may not involve transcriptional control.

Sugars Alter mRNA Stability

To examine the effect of sugars on the mRNAhalf-life of individual genes, degradation of mRNA

in vivo was monitored by RNA gel-blot analysisfollowing the inhibition of transcription with actino-mycin D (Act D). Addition of 10 mg mL21 Act D tothe medium inhibits total RNA transcription by morethan 95% over a 12-h time-course (Sheu et al., 1994).To obtain a high starting level of mRNA for detectionof changes in mRNA accumulation, cells were cul-tured with Suc for SU genes (Fig. 4a), or without Sucfor SD genes (Fig. 4b), prior to subjecting cells toinhibition of transcription with Act D. Whereas mea-surement of mRNA half-lives following differentgrowth regimes makes it difficult to directly comparethe absolute mRNA half-lives between SU and SDgenes, the relative mRNA half-lives of the samegenes under starved or non-starved conditions can becompared. Using the latter method, we previouslydetected the effect of sugars on a-amylase mRNAhalf-lives (Sheu et al., 1994, 1996) and confirmed therole of aAmy3 39-UTR on sugar-dependent mRNAstability (Chan and Yu, 1998a, 1998b).

In the present study, mRNA levels of all the SUgenes were relatively low in Suc-starved cells (Fig.4a, lane 1) but high in Suc-provided cells (Fig. 4a,lane 2). After cells were pretreated with Act D plus

Figure 2. Alterations in protein levels by sugars parallel alterations inmRNA levels. Rice suspension cells were cultured in Suc-containing(1S) medium for 72 h, transferred to Suc-free (2S) medium for 72 h,and transferred to 1S medium for an additional 72 h. Total proteinswere extracted from rice suspension cells and subjected to proteingel-blot analysis. The amount of total proteins applied in each lanewas 10 mg for a-Amy and 50 mg for Act or SSP2.

Figure 3. Sugars affect transcription rates of sugar-regulated genes.Rice suspension cells were grown in Suc-containing (1S) medium orSuc-free (2S) medium for 24 h. Cells were collected and nuclei wereisolated. Run-on transcription analysis was performed as previouslydescribed (Sheu et al., 1994). The indicated DNA was isolated fromplasmids as described in “Materials and Methods.” Equal moleculesof indicated DNA were slot-blotted on the membrane. a, Transcrip-tion of SU genes. b, Transcription of SD genes. pBS, pBluescriptDNA. The blots were visualized with autoradiography and quantifiedusing phosphor-imaging software. Numbers below the autoradio-graphs of individual genes indicate the ratio of transcription ratesbetween 1S and 2S cells. The experiment was repeated twice withsimilar results.

Ho et al.


Dow



ovember 2021

Suc for 12 h, levels of Act, ADH2, G3PD, HSP86, andSSP2 mRNA decreased more slowly in cells providedwith ActD plus Suc (Fig. 4a, lanes 3–11) than in cellsprovided with ActD minus Suc (Fig. 4a, lanes 12–20)during the next 12 h. The amounts of mRNA (Fig. 4a)were quantified using phosphor-imaging software(PhosphorImager, Molecular Dynamics, Sunnyvale,CA) and mRNA half-lives were determined (Table I).The half-lives of Act, ADH2, G3PD, and SSP2mRNAs were approximately 1.6- to 2.6-fold longer inSuc-provided cells than in Suc-starved cells. The half-lives of His and HSP86 mRNAs were not signifi-cantly altered by varying Suc levels.

For all the tested SD genes, the mRNA levels wererelatively low in Suc-provided cells (Fig. 4b, lane 1)and significantly higher in Suc-starved cells (Fig. 4b,lanes 2–4). After cells were pretreated with Act Dminus Suc for 12 h, the mRNA levels decreased morerapidly in cells provided with ActD plus Suc (Fig. 4b,lanes 5–12) than in cells provided with ActD minusSuc (Fig. 4b, lanes 13–19) during the next 9 h. Thehalf-life of a-Amy mRNA was 11.0-fold longer inSuc-starved cells than in Suc-provided cells (TableII), which was similar to our previous findings (Sheuet al., 1994). The half-lives of other mRNAs were 2.5-to 7.4-fold longer in Suc-starved cells than in Suc-provided cells (Table II). The above results demon-strate that mRNA stability plays an important role inthe expression of some SU and all SD genes.

Phosphorylation of Hexose Activates or SuppressesSugar-Regulated Gene Expression

We previously showed that expression of a-amylase genes in rice suspension cells was sup-Figure 4. Sugars alter mRNA stability of sugar-regulated genes. a,

Measurement of mRNA half-life of SU genes. Rice suspension cellswere cultured in Suc-free (2S) medium for 24 h (RNA in lane 1) or inSuc-containing (1S) medium for 24 h to increase the starting RNAlevel (RNA in lane 2). ActD was then added to the 1S medium to afinal concentration of 10 mg/mL and cells were incubated for another12 h and then divided in half. One-half of the cells were transferredto a 1S medium containing ActD (RNAs in lanes 3–11). The otherone-half were transferred to a 2S medium containing ActD (RNAs inlanes 12–20). Cells were incubated and collected from 0 to 12 h andRNA was purified. RNA gel-blot analysis was performed using probesof SU genes. Ten micrograms of total RNA were loaded in each lane.Three membrane blots were parallel prepared. The Act, SSP2 andrRNA probes, ADH2 and HSP86 probes, and G3PD and His probeswere respectively hybridized to each of the three blots. b, Measure-ment of mRNA half life of SD genes. Rice suspension cells werecultured in 1S medium for 24 h (RNA in lane 1) and transferred to 2S

Table I. mRNA half lives of Suc up-regulated genes

Gene/EnzymeHalf Life (h) Fold of Change in

Half Life (1S/2S)1S 2S

Act 13.2 5.0 2.6ADH2 9.9 5.5 1.8His 14.0 12.0 1.2G3PD 14.6 9.2 1.6HSP86 10.6 9.1 1.2SSP2 9.2 4.9 1.9

medium for 24 h to increase the starting RNA level (RNA in lane 2).ActD was then added to 2S medium to a final concentration of 10mg/mL and cells were incubated for another 12 h and then divided inhalf. One-half of the cells were transferred to a 1S medium contain-ing ActD (RNAs in lanes 5–12). The other one-half were transferredto a 2S medium containing ActD (RNAs in lanes 13–19). Cells wereincubated and collected from 0 to 9 h and RNA was purified. RNAgel-blot analysis was performed using probes of SD genes. Twomembrane blots were parallel prepared and sequentially hybridizedwith these probes. The a-Amy probe hybridized to one blot and therest of the probes hybridized to another blot. Ten micrograms of totalRNA were loaded in each lane. Lanes 3 and 4, Cells were incubatedin 2S medium lacking ActD for 36 and 45 h, respectively, to serve ascontrols.



Dow



ovember 2021

pressed by the presence of Glc, Fru, or Suc in theculture medium, and Suc was hydrolyzed to Glc andFru by the cell wall invertase prior to uptake by ricecells (Yu et al., 1991). The phosphorylation of hexosesugars by hexokinase has been shown to be critical forrepression of photosynthetic gene expression in maizeprotoplasts (Jang and Sheen, 1994) and glyoxylate cy-cle gene expression in cucumber cell culture (Grahamet al., 1994). To investigate whether phosphorylationof hexose sugars activates or suppresses sugar-regulated gene expression, we conducted experimentsusing Glc analogs. Rice suspension cells were culturedin 3-O-methyl-Glc (3-OMG) and 6-deoxy-Glc (6-dG),which are taken up by cells but are not phosphory-lated by hexokinase, and also 2-deoxy-Glc (2-dG),which is phosphorylated by hexokinase but is notfurther metabolized by cells (Dixon and Webb, 1979).Application of 2-dG at various effective concentrations(as indicated by suppression of a-amylase gene ex-pression) between 0.1 to 0.5 mm, which were belowthe commonly used concentrations for plant cells(Graham et al., 1994; Jang and Sheen, 1994), resulted ina toxic effect on cell growth (as indicated by abnormalmorphology of suspension cells and increased degra-dation of rRNA) within 12 h. The data of treatmentwith 2-dG were therefore not presented in this report.The effects of 40 mm each of Suc, Glc, 3-OMG, and6-dG on steady-state mRNA levels of SU and SD genesare shown in Figure 5. mRNA levels of all the SUgenes were relatively high in cells grown in mediumcontaining Suc or Glc (Fig. 5a, lanes 2–5), but weresignificantly lower in cells grown in medium lackingsugar or containing 3-OMG or 6-dG for 12 h (Fig. 5a,lanes 7, 9, and 11). In contrast, mRNA levels of all theSD genes as well as aAmy3 were relatively low orbarely detectable in cells grown in medium containingSuc or Glc (Fig. 5b, lanes 2–5) but were significantlyhigher in cells grown in medium lacking sugar orcontaining 3-OMG or 6-dG for 12 h (Fig. 5b, lanes 7, 9,and 11).

Because 2-dG was toxic to plant cells, rice suspen-sion cells were cultured in another Glc analog, Man.Upon entry into the plant cell, Man is phosphory-lated by hexokinase, yielding Man-6-P. Man-6-P isthen slowly processed by plant cells, since the en-zymes required for this process are either absent orexist in very low concentrations (Walder and Sivak,1986). Man has been shown to inhibit germination of

Arabidopsis seeds due to phosphorylation by hexoki-nase and not due to ATP or P depletion (Peto et al.,1999). As shown in Figure 6, the effect of Glc onmRNA accumulation of SU and SD genes was similaras in Figure 5. Man slightly activated the expressionof ADH2, G3PD, and HSP86 genes but not that of Actand SSP2 genes in sugar-starved cells (Fig. 6a, com-pare lane 4 with lane 6). In contrast, Man suppressedthe expression of aAmy3, aAmy8, and A3 but not thatof A2, B1, D1, E1, and F1 in sugar-starved cells (Fig.6b, compare lane 4 with lane 6).

The above results demonstrate that Glc by itself orGlc and Fru hydrolyzed from Suc all can activate theexpression of SU genes and suppress the expressionof SD genes. In contrast, the Glc analogs, 3-OMG and6-dG, which cannot be phosphorylated and metabo-lized by cells, do not activate the expression of SUgenes or suppress the expression of SD genes. TheGlc analog, Man, which can be phosphorylated butslow metabolized by cells, partially activates the ex-pression of only certain SU genes. Man behaves in amixed fashion with respect to SD genes by suppress-ing or activating expression of different SD genes.

The Metabolic Intermediate Pyruvate SuppressesGene Expression

Pyruvate has been shown to prevent autophage incarbohydrate-starved sycamore cells, and since pyru-vate is the main mitochondrial substrate derivedfrom glycolysis, it was suggested that pyruvate pre-vents autophage by supplying the mitochondria witha respiratory substrate (Aubert et al., 1996). Pyruvatewas tested for its ability to activate or suppresssugar-regulated gene expression. Pyruvate did notactivate the expression of all the SU genes in Suc-starved cells (Fig. 7a, compare lane 4 with lane 8),however, it enhanced the expression of G3PD andSSP2 in Suc-provided cells (Fig. 7a, compare lane 2with lane 6). On the other hand, pyruvate suppressedthe expression of all the SD genes, except aAmy3 andA3, in Suc-starved cell (Fig. 7b, compare lane 4 withlane 8). It is interesting that pyruvate enhanced theexpression of aAmy3 and A3 in Suc-starved cells (Fig.7b, compare lane 4 with lane 8). These results dem-onstrate that expression of certain SD genes can besuppressed by the metabolic intermediate pyruvate.

DISCUSSION

Growth-Related Genes Are Positively But May or MayNot Be Coordinately Regulated by Sugars

In the present study, preferential expression ofgrowth-related genes in Suc-provided growing cellswas as expected (Fig. 1). The parallel change in levelsof a-Amy, Act, and SSP2 proteins (Fig. 2) with theirmRNAs in response to sugar availability suggeststhat these sugar-responsive proteins may be physio-

Table II. mRNA half lives of Suc down-regulated genes

Gene/EnzymeHalf Life (h) Fold of Change in

Half Life (2S/1S)2S 1S

a-Amy 13.2 1.2 11.0A2 10.2 3.4 3.0A3 32.0 4.3 7.4B1 23.6 5.2 4.5D1 12.2 4.8 2.5E1 14.2 2.9 4.9F1 11.9 4.0 3.0

Ho et al.


Dow



ovember 2021

logically relevant. The expression of most SU genes iscoordinately up-regulated by sugars. However, ex-pression of His, HSP86, and Ubi genes may also benon-coordinately regulated, probably depending onthe special needs of different members of these pro-tein families under various growth conditions. Thepresent study did not focus on monitoring the ex-pression of various genes in response to varyingsugar levels based on gene-specific probes; a singlegene or distinct groups of genes may contribute tothe overall expression pattern. Histones are requiredin large quantities at the time of cell division, and inmany eukaryotic systems histones are synthesizedprimarily during the S phase of the cell cycle (Geth-ing and Sambrook, 1992). The quick and dramatic buttransient increase in the expression of His 1 h aftershifting cells from 2S to 1S medium (Fig. 1a, lane 15)suggests a burst in DNA replication after resumptionof sugar supply. Heat shock proteins (HSPs) are as-sumed to participate in the maintenance of cellularstructure during heat stress as well as to act as mo-lecular chaperones in normally growing cells(Viestra, 1996). This may explain the phenomenonthat the expression of HSPs was high in Suc-providedcells (Fig. 1a, lanes 1–4), decreased initially after Sucstarvation (Fig. 1a, lanes 5–9), but increased again atlater stages of starvation (Fig. 1a, lanes 10–14). Ubiq-uitin is responsible for cellular housekeeping as wellas stress response by removing abnormal or mis-folded proteins and recycling amino acids (Mikamiand Iwabuchi, 1993). This may explain the contrast-ing expression patterns of different members of ubiq-uitin genes in response to the availability of sugars(Fig. 1a).

Stress-Related Genes Are Coordinately and NegativelyRegulated by Sugars

The Suc-repressible genes identified in this study,except for a-amylase genes, all encode stress-relatedproteins and represent a group of genes distinct fromthose previously identified as being involved in pho-tosynthesis and reserve remobilization and catabo-lism (Koch, 1996). cDNA B1 encodes a Gly-rich pro-tein (GRP) that shows homology to RNA-bindingproteins containing the ribonucleoprotein consensus

Figure 5. Non-phosphorylatable Glc analogs do not activate or re-press sugar-responsive gene expression. Rice suspension cells werecultured in Suc-containing medium for 5 d and transferred to me-dium containing 40 mM Suc (S), 40 mM Glc (G), no sugar (2), 40 mM

3-OMG, or 10 mM 6-dG for 2 and 12 h. Total RNA was purified andsubjected to RNA gel-blot analysis. a, SU genes as probes. b, SDgenes as probes. Four membrane blots were parallel prepared. TheSSP2, rDNA and E1 probes, ADH2, HSP86, aAmy3, and D1 probes,Act, G3PD, A3, and B1 probes, A2 and F1 probes were respectivelyhybridized to each of the four blots.



Dow



ovember 2021

sequence. A class of RNA-binding GRP genes re-sponsive to wounding and cold were identified inplants (Sturm, 1992; Horvath and Olson, 1998).cDNAs A2, D1, E1, and F1 encode GRPs that showhomology to a group of Gly-rich cell wall or periplas-mic space-associated proteins (Fang et al., 1991; Leiand Wu, 1991). GRPs are widely distributed in plantsand are developmentally regulated (Cheng et al.,1996) and responsive to wounding, virus infection,drought, and flooding (Condit and Meagher, 1987;Gomez et al., 1988; de Oliveira et al., 1990; Fang et al.,1991). cDNA A3 is almost identical to the salt-responsive rice gene salT with only minor nucleotidevariation. Expression of salT is significantly inducedin sheaths and roots after plants are osmotically chal-lenged (Claes et al., 1990) and in cultured rice sus-pension cells by abscisic acid and NaCl (Garcia et al.,1998).

Suc starvation is a type of nutritional stress. Coor-dinated expression of GRP genes and salT in Suc-starved cells suggests that these genes may play rolesin protecting cells against nutritional stress. In addi-tion, inducibility of expression of these genes in ricecell culture by sugar starvation also suggests thatdifferent stress conditions might trigger a generalresponse by creating the same intracellular signal.The inducibility of expression of various stress-related genes in a rice cell culture by sugar starvationmakes the rice cell culture an ideal model system forstudying the signal transduction pathway and regu-latory mechanism of plant stress response.

Dual Regulation by Sugars Operates atTranscriptional and Posttranscriptional Levels

We found that sugars not only regulates the tran-scription but also alters the stability of mRNAs.Among the six SU genes tested, under the experimen-tal condition in which the transcription rate was mea-sured and compared 24 h after cell culture with orwithout Suc, an increase in transcription rate in Suc-provided cells was observed only for ADH2 andG3PD (Fig. 3a). At the same time, accumulation ofHSP86 mRNA in Suc-starved cells began to rise (Fig.1a, lane 11). This may explain why at this time pointthe transcription rate of HSP86 gene was higher inSuc-starved cells than in Suc-provided cells (Fig. 1a,slot 2). Since the mRNA half-life of HSP86 mRNAwas not significantly altered by Suc level (Table I),

Figure 6. Phosphorylatable but slowly metabolized Glc analog ac-tivates or represses certain gene expression. Rice suspension cellswere cultured in Suc-containing medium for 5 d and transferred tomedium containing 40 mM Glc (1G), no Glc (2G), or 2 mM Man for2 and 12 h. Total RNA was purified and subjected to RNA gel-blotanalysis. a, SU genes as probes. b, SD genes as probes. Six membraneblots were parallel prepared. The Act, HSP86 and rDNA probes,ADH2, G3PD, and SSP2 probes, aAmy3 and D1 probes, aAmy8 andB1 probes, A3 and F1 probes, and A2 probe, were respectivelyhybridized to each of the six blots.

Ho et al.


Dow



ovember 2021

transcriptional activity may play a more dominantrole than mRNA stability in the accumulation ofHSP86 mRNA. The transcription rate of the SSP2gene was similar in either Suc-provided or Suc-starved cells and the transcription rate of the Actgene was lower in Suc-provided cells than in Suc-starved cells (Fig. 3a). These findings suggest thatmRNA stability may play a more dominant role thantranscriptional activity in accumulation of these twomRNAs in Suc-provided cells. This notion is sup-ported by the observation that the half lives of Actand SSP2 mRNAs are 2- to 3-fold longer in Suc-provided cells than in Suc-starved cells (Table I). Themassive and transient accumulation of His mRNA1 h after shifting cells from 2S to 1S medium (Fig.1a, lane 15) suggests a sugar hypersensitive but shorthalf-lived increase in transcriptional activity and/ormRNA stability coupled with His DNA synthesisduring a short time window. The half-lives of ADH2and G3PD are 1.6- to 1.8-fold longer in Suc-providedcells than in Suc-starved cells. Therefore, in thepresent study, sugar up-regulation of transcriptionand mRNA stability are coupled only for ADH2,G3PD, and His. On the other hand, sugar down-regulation of transcription and mRNA stability arecoupled for all of the SD genes.

Sugars Regulate Gene Expression through CommonControl Mechanisms

The coordinated activation or repression of sugar-responsive genes suggests that common mechanismsmight be responsible for regulation of these genes.The mechanisms would include interaction betweentransacting factors (transcription or RNA degrada-tion factors) and cis-regulatory elements (promoteror mRNA sequences) of the individual genes. Spe-cific cis-regulatory elements in the promoters or mR-NAs are probably shared by these sugar-regulatedgenes. The SRC of aAmy3 promoter contains aTATCCA element that has been shown to serve as anenhancer for sugar starvation-induced expression(Lu et al., 1998). The TATCCA element along with itsvariants are present at a proximity upstream of thetranscription start sites of 18 a-amylase genes iso-lated from various plant species (Yu, 1999b) andother sugar-repressible genes, e.g. the cucumbermalate synthase and isocitrate lyase genes (Grahamet al., 1989; Reynolds and Smith, 1995) and the maizeSuc synthase gene (Sh1) (Koch et al., 1992). The pro-

Figure 7. Pyruvate represses SD gene expression. Rice suspensioncells were cultured in Suc-containing (1S) medium for 5 d andtransferred to 1S medium or Suc-free (2S) medium with or without50 mM pyruvate. Total RNA was purified and subjected to RNAgel-blot analysis. a, SU genes as probes. b, SD genes as probes. Sixmembrane-blots were parallel prepared. The HSP86, aAmy3 andaAmy8 probes, SSP2 and A3 probes, Act and F1 probes, G3PD, B1,and E1 probes, rRNA and D1 probes, ADH2 and A2 probes wererespectively hybridized to each of the six blots.



Dow



ovember 2021

moter region of Sh1 containing the TATCCA elementalso confers sugar inhibition of downstream reportergene expression (Maas et al., 1990). These findingssuggests that the TATCCA element or its variantscould be a common cis-regulatory element responsi-ble for sugar repression. Computer analysis foundthat nucleotide sequences similar to the essential mo-tifs in the aAmy3 39-UTR for sugar-dependent mRNAstability (Chan and Yu, 1998a, 1998b) were alsopresent in the 39-UTRs of the SD genes examined inthe present study. Whether these mRNA sequencesare functional remain to be determined. Promoteranalysis of the sugar up-regulated patatin class-Igene identified two separate cis-sequences responsi-ble for sugar induction (Grierson et al., 1994). Thecis-sequences contain two conserved 9-bp Suc re-sponse elements (SURE), which are similar to somemotifs present in the promoters of some SU genes,e.g. the b-amylase and sporamin genes of sweet po-tato (Ohta et al., 1991; Ishiguro and Nakamura, 1992)and the proteinase inhibitor II gene of potato (Kim etal., 1991). In future studies identification of genesencoding the transacting transcription factors wouldallow us to understand whether a common or mul-tiple transcription factors interact with the cis-regulatory elements and are involved in the coordi-nated transcription of SU or SD genes.

Hexokinase-Dependent and -Independent PathwaysRegulate Gene Expression and the Sugar SignalTransduction Pathway May Overlap the GlycolyticPathway for Gene Repression

Various glycolytic intermediates downstream ofGlc have been shown to have no repressive effect onphotosynthetic gene expression (Jang and Sheen,1994). In the present study, pyruvate, which is pro-duced at the final step of glycolysis, suppressed theexpression of all the SD genes except aAmy3 and A3in Suc-starved cells. This study provides the firstevidence that the sugar signal transduction pathwaymay overlap the downstream sugar metabolic path-way for gene regulation.

Recent study has indicated that sugar signaling inplants occurs by hexokinase-dependent pathway, byhexose-dependent but hexokinase-independent path-way, and by Suc-dependent pathway (Jang andSheen, 1997; Smeekens and Rook, 1997; Halford et al.,1999; Sheen et al., 1999; Smeekens, 2000). Our obser-vation is that Glc and Suc are equally effective in up-and down-regulation of SU and SD gene expression,respectively, suggesting that in the present study thefirst two pathways may play more important roles inthe activation and repression of the SU and SD genes,respectively. Man slightly activates the expression ofADH2, G3PD, and HSP86 genes but not that of Actand SSP2 genes (Fig. 6a). Pyruvate did not activatethe expression of all the SU genes (Fig. 7a). Theseresults suggest that hexokinase may play a signaling

role in activation of ADH2, G3PD, and HSP86 geneexpression, and hexose-dependent but hexokinase-independent pathway activates the expression of Actand SSP2 genes. On the other hand, Man suppressedthe expression of aAmy3, aAmy8, and A3 but not thatof A2, B1, D1, E1, and F1 (Fig. 6b). Pyruvate sup-presses the expression of aAmy8, A2, B1, D1, E1, andF1 but not that of aAmy3 and A3. These resultssuggest that hexokinase may play a signaling role insuppression of aAmy3, aAmy8, and A3, and the met-abolic pathway delivers signal to suppress expres-sion of aAmy8, A2, B1, D1, E1, and F1. Figure 8asummarizes the results of Figures 6 and 7 and showsthat although coordinately regulated by sugars, dif-ferent genes are up-regulated or down-regulatedthrough hexokinase-dependent and/or hexokinase-

Figure 8. Complex sugar signal transduction networks for differentialand coordinated regulation of gene expression. a, Signal transductionin sugar-provided cells. Suc may be sensed by the receptor directly,or it may be converted to Glc and Fru and sensed by hexokinase-dependent or hexokinase-independent pathway. The sugar signaltransduction pathway may overlap or be independent of the sugarmetabolic pathway. b, Signal transduction in Suc-starved cells. Thesignals of sugar starvation and environmental stresses may be per-ceived by the same receptor or by different receptors but the signalpathways converge downstream. R, Receptor; INV, invertase. Thesolid arrows indicate pathways proposed based upon currently avail-able information. 1, 2; Positive and negative regulation, respectively.

Ho et al.


Dow



ovember 2021

independent pathways. In addition, the sugar signaltransduction pathway may overlap the glycolyticpathway for gene repression.

Convergence of Nutritional and Environmental StressSignal Transduction Pathways

Complex regulatory circuitries may also link thesugar starvation signal to the stress signals (Fig. 8b)for differential and coordinated regulation of geneexpression. In sugar-starved cells, as expression ofa-amylase and stress-related genes are coordinatelyregulated, the signals of sugar starvation and envi-ronmental stresses may be perceived by the samereceptor, or by different receptors while the signalpathways converge downstream (Fig. 8b). The latterpathways have been proposed in a study on sugar-and stress-regulated sink-specific and defense-related gene expression in Chenopodium rubrum(Ehness et al., 1997). Under either condition, the exactpoints where the stimulating and inhibitory path-ways diverge from the same signal transductionchains are not known. Whether the signals of sugarand sugar starvation are transduced through inde-pendent pathways or through the opposite action ofthe same components in the signal transductionchain is also not known.

Although in the present study the stress-relatedgenes are randomly selected and the mode of theirregulation by sugars may not be applied to all stress-related gene, the requirement of convergence ofsugar starvation and stress signal transduction path-ways could be of physiological significance. In stresssituations, cells have a high demand for sugars tofulfil the energy and carbon requirements needed forappropriate response to stresses. This may explainwhy expression of a-amylases, which hydrolyzestarch to provide sugars for metabolic activity, cou-ples with the expression of stress-related proteins.The notion is supported by observations that expres-sion of a-amylases is induced in barley leaves bywater stress (Jacobsen et al., 1986), in tobacco leavesby virus infection (Heitz et al., 1991), and in mungbean cotyledons by wounding (Koizuka et al., 1995).Similar phenomena are found for other hydrolyticenzymes. For example, expression of b-fructosidase(invertase), which hydrolyzes stored Suc to hexose, isenhanced in carrot roots in response to wounding orpathogen infection (Sturm and Chrispeels, 1990). Theincrease in expression of these carbohydrate hydro-lytic enzymes suggests a role of these enzymes indefense responses to biotic or abiotic stress.

Among the SD genes, A2, B1, D1, E1, and F1 seemto be controlled by sugar via a similar or identicalpathway, whereas A3 is controlled via somewhatdifferent pathways (Fig. 8a). The notion that expres-sion of aAmy3 and aAmy8 are also differentially reg-ulated via somewhat different pathways is supportedby the observation that pyruvate suppressed the ex-pression of aAmy8 but not aAmy3 (Figs. 7b and 8a).

In conclusion, sugars can modulate gene expres-sion at the transcriptional and posttranscriptionallevels through a complex signal transduction net-work and some common mechanisms. Additionalwork is necessary to identify the various componentsof the signal transduction chains and to explore howthe interaction between the cis-acting elements in thepromoters and/or mRNAs with the trans-acting fac-tors are involved in the coordinated but differentialregulation of diverse gene expression in response tothe sugar status in plant cells. Such future studiesshould lead to a better understanding of the mecha-nisms that underlie the global sugar regulation ofgene expression in plants.

MATERIALS AND METHODS

Rice (Oryza sativa cv Tainan 5) Cell Culture

A suspension cell culture of rice was established aspreviously described (Yu et al., 1991). Cells were subcul-tured every 7 d by transferring approximately 0.5 mL ofcells into 25 mL of fresh liquid Murashige and Skoogmedium (Murashige and Skoog, 1962) containing 3% (w/v)Suc in a 125-mL flask. Cells were cultured on a reciprocalshaker at 120 rpm and incubated at 26°C in the dark.

Plasmids

Plasmid aAmy8-C carries a 1.4-kb rice a-amylase cDNAinsert in pBluescript KS1 (Stratagene, La Jolla, CA) (Yu etal., 1992). Plasmid pcRAc1.3 contains a 1.4-kb rice actin 1cDNA insert in pBluescript II-KS (McElroy et al., 1990).Plasmid pRY18 carries a 3.8-kb DNA fragment that con-tains a rice genomic rDNA cluster, including the 39-halfportion of 18S rRNA gene, the complete 5.8S rRNA gene,and the 59-half portion of the 25S rRNA gene in pUC13(Sano and Sano, 1990). cDNAs encoding ADH2, G3PD,HSP86, His, SSP2, and Ubi were isolated from rice calluscDNA libraries and inserted in SalI(59) and NotI(39) sites ofpBluescript SKII1 (Stratagene) by the Japan Rice GenomeResearch Program (Uchimiya et al., 1992).

RNA Gel-Blot Analysis

Total RNA was purified from rice suspension cells usingTRIZOL reagent (Life Technologies/Gibco-BRL, Cleve-land). a-32P-labeled DNA probes were prepared and RNAgel-blot analysis was performed as described (Sheu et al.,1996). In cases when the membrane blot was sequentiallyhybridized with various probes, each probe on the mem-brane was stripped and rehybridized as described (Sheu etal., 1994). Plasmid DNAs of aAmy8-C and pcRAc1.3 weredigested with EcoRI. Plasmid DNAs of Act, ADH2, G3PD,His, HSP86, and SSP2 were digested with SalI and NotI.Plasmid DNAs of A1, A3, B1, D1, E1, and F1 were digestedwith EcoRI. aAmy3 and aAmy8 gene-specific DNAs wereprepared as described (Sheu et al., 1996). The insert DNAswere individually isolated, labeled with 32P, and used asprobes. A DNA fragment containing 25S, 18S, and 5.8S



Dow



ovember 2021

rDNAs was excised from pRY18 using BamHI, labeled witha-32P, and used as a probe for equalizing RNA loading.

Differential Screening of cDNA Library

Rice suspension cells were cultured in Suc-containingmedium for 5 d and transferred to Suc-containing (1S) orSuc-free (2S) medium for 4 h. Cells were collected andtotal RNA was purified. Poly(A)1 RNA was further puri-fied using an oligo(dT)-cellulose spin column (593 39). Thepoly(A)1 RNA isolated from 2S cells was used to constructa cDNA library in lGEM-2 vector (Promega, Madison, WI).The 32P-labeled single-stranded cDNA probe was preparedfrom poly(A)1 RNA of 1S or 2S cells using an oligo(dT)primer and avian myeloblastosis virus reverse transcrip-tase. Duplicated filter lifts from high-density platings of thecDNA library were then differentially screened with thecDNA probes. The phage plaques that hybridized stronglywith the cDNA probe of 2S cells and only weakly or not atall with the cDNA probe of 1S cells were isolated. ThecDNA in lGEM-2 was then cleaved with EcoRI, subclonedinto the EcoRI site of pBluescript vector, and nucleotidesequenced.

Protein Gel-Blot Analysis

Total proteins were extracted from cultured suspensioncells with an extraction buffer (50 mm Tris [tris(hydroxy-methyl)aminomethane]-HCl, pH 8.8, 1 mm EDTA, 10%[v/v] glycerol, 1% [v/v] Triton X-100, 10 mm b-mercapto-ethanol, and 0.1% [w/v] sarkosyl). The culture medium wascollected and centrifuged at 18,000g at 4°C for 15 min toremove cell debris. Protein gel-blot analysis was performedas described (Yu et al., 1991). The anti-a-amylase polyclonalantibodies raised against rice a-amylases were previouslyproduced in our laboratory (Chen et al., 1994). The anti-Sucsynthase polyclonal antibodies were raised in rabbit againstSuc synthase purified from developing rice grains and werea gift from Ai-Yu Wang (National Taiwan University, Tai-pei). The mouse anti-actin monoclonal antibody raisedagainst chicken gizzard actin was purchased from ChemiconInternational, Inc.

ACKNOWLEDGMENTS

We thank Dr. Jun-Jei Sheu and Ms. Lin-Tze Yu for tech-nical assistance and Mr. Douglas Platt for help in preparingthe manuscript. We also thank the Japan Rice GenomeResearch Program for providing us the rice cDNAs.

Received June 6, 2000; returned for revision August 25,2000; accepted October 7, 2000.

LITERATURE CITED

Aubert S, Gout E, Bligny R, Marty-Mazars D, Barrieu F,Alabouvette J, Marty F, Douce R (1996) Ultrastructuraland biochemical characterization of autophagy in higherplant cells subjected to carbon deprivation: control by the

supply of mitochondria with respiratory substrates.J Cell Biol 133: 1251–1263

Brouquisse R, James F, Pradet A, Raymond P (1992) As-paragine metabolism and nitrogen distribution duringprotein degradation in sugar-starved maize root tips.Planta 188: 384–395

Carlson M (1987) Regulation of sugar utilization in Saccha-romyces species. J Bacteriol 169: 4873–4877

Chan M-T, Chao Y-C, Yu S-M (1994) Novel gene expres-sion system for plant cells based on induction ofa-amylase promoter by carbohydrate starvation. J BiolChem 269: 17635–17641

Chan M-T, Yu S-M (1998a) The 39-untranslated region of arice a-amylase gene mediates sugar-dependent abun-dance of mRNA. Plant J 15: 685–696

Chan M-T, Yu S-M (1998b) The 39-untranslated region of arice a-amylase gene functions as a sugar-dependentmRNA stability determinant. Proc Natl Acad Sci USA 95:6543–6547

Chen M-H, Liu L-F, Chen Y-R, Wu H-K, Yu S-M (1994)Expression of a-amylases, carbohydrate metabolism, andautophagy in cultured rice cells are coordinately regu-lated by sugar nutrient. Plant J 6: 625–636

Cheng S-H, Keller B, Condit CM (1996) Common occur-rence of homologues of petunia glycine-rich protein-1among plants. Plant Mol Biol 31: 163–168

Cheng W-H, Taliercio EW, Chourey PS (1999) Sugarsmodulate an unusual mode of control of the cell-wallinvertase gene (Incw1) through its 39-untranslated regionin a cell suspension culture of maize. Proc Natl Acad SciUSA 96: 10512–10517

Chevalier C, Bourgeois E, Pradet A, Raymond P (1995)Molecular cloning and characterization of six cDNAsexpressed during glucose starvation in excised maize(Zea Mays L.) root tips. Plant Mol Biol 28: 473–485

Claes B, Dekeyser R, Villarroel R, Van den Bulcke M,Bauw G, Van Montagu M, Caplan A (1990) Character-ization of a rice gene showing organ-specific expressionin response to salt stress and drought. Plant Cell 2: 19–27

Condit CM, Meagher RB (1987) Expression of a gene en-coding a glycine-rich protein in petunia. Mol Cell Biol 7:4273–4279

de Oliveira DE, Seurinck J, Inze D, Van Montagu M,Botterman J (1990) Differential expression of five Arabi-dopsis genes encoding glycine-rich proteins. Plant Cell 2:427–436

DeWald DB, Sadka A, Mullet JE (1994) Sucrose modula-tion of soybean Vsp gene expression is inhibited byauxin. Plant Physiol 104: 439–444

Dixon M, Webb EC, eds (1979) Enzymes. Longman Scien-tific & Technical, London, pp 248–251

Ehness R, Ecker M, Godt DE, Roitsch T (1997) Glucoseand stress independently regulate source and sink me-tabolism and defense mechanisms via signal transduc-tion pathways involving protein phosphorylation. PlantCell 9: 1825–1841

Efrat S, Tal M, Lodish HF (1994) The pancreatic b-cellglucose sensor. Trends Biochem Sci 19: 535–538

Ho et al.


Dow



ovember 2021

Entian K-D, Barnett JA (1992) Regulation of sugar utiliza-tion by Saccharomyces cerevisiae. Trends Biochem Sci 17:506–510

Fang R-X, Pang Z, Gao D-M, Mang K-Q, Chua N-H (1991)cDNA sequence of a virus-inducible, glycine-rich proteingene from rice. Plant Mol Biol 17: 1255–1257

Gancedo JM (1992) Carbon catabolite repression in yeast.Eur J Biochem 206: 297–313

Garcia AB, de Almeida Engler J, Claes B, Villarroel R,Van Montagu M, Gerats T, Caplan A (1998) The expres-sion of the salt-responsive gene salT from rice is regu-lated by hormonal and developmental cues. Planta 207:172–180

Gething MJ, Sambrook J (1992) Protein folding in the cell.Nature 355: 33–45

Gomez J, Sanchez-Martinez D, Stiefel R, Rigau J, Puig-domenech P, Pages M (1988) A gene induced by theplant hormone abscisic acid in response to water stressencodes a glycine-rich protein. Nature 334: 262–264

Graham IA, Denby KJ, Leaver CJ (1994) Carbon cataboliterepression regulates glyoxylate cycle gene expression incucumber. Plant Cell 6: 761–772

Graham IA, Smith LM, Brown JW, Leaver DJ, Smith SM(1989) The malate synthase gene of cucumber. Plant MolBiol 13: 673–684

Grierson C, Du J-S, de Torres Zabala M, Beggs K, SmithC, Holdsworth M, Bevan M (1994) Separate cis se-quences and trans factors direct metabolic and develop-mental regulation of a potato tuber storage protein gene.Plant J 5: 815–826

Halford NG, Purcell PC, Grahame Hardie D (1999) Ishexokinase really a sugar sensor in plants. Trends PlantSci 4: 117–120

Hattori T, Nakagawa S, Nakamura K (1990) High levelexpression of tuberous root storage protein genes ofsweet potato in stems of plantlets grown in vitro onsucrose medium. Plant Mol Biol 14: 595–604

Heitz T, Gioffroy P, Fritig B, Legrand M (1991) Twoapoplastic a-amylases are induced in tobacco by virusinfection. Plant Physiol 97: 651–656

Herbers K, Meuwly P, Frommer WB, Metraux J-P, Son-newald U (1996) Systemic acquired resistance mediatedby the ectopic expression of invertase: possible hexosesensing in the secretory pathway. Plant Cell 8: 793–803

Horvath DP, Olson PA (1998) Cloning and characteriza-tion of cold-regulated glycine-rich RNA-binding proteingenes from leafy spurge (Euphorbia esula L.) and compar-ison to heterologous genomic clones. Plant Mol Biol 38:531–538

Ishiguro S, Nakamura K (1992) The nuclear factor SP8BFbinds to the 59-upstream regions of three different genescoding for major proteins of sweet potato tuberous roots.Plant Mol Biol 14: 995–1006

Jacobsen JV, Hanson AD, Chandler PC (1986) Water stressenhances expression of an a-amylase gene in barleyleaves. Plant Physiol 80: 350–359

James F, Brouquisse R, Pradet A, Raymond P (1993)Changes in proteolytic activities in glucose-starvedmaize root tips: regulation by sugars. Plant Physiol Bio-chem 31: 845–856

Jang J-C, Leo P, Zhou L, Sheen J (1997) Hexokinase as asugar sensor in higher plants. Plant Cell 9: 5–19

Jang J-C, Sheen J (1994) Sugar sensing in higher plants.Plant Cell 6: 1665–1679

Jang J-C, Sheen J (1997) Sugar sensing in higher plants.Trends Plant Sci 2: 208–213

Jefferson R, Goldsbrough A, Bevan M (1990) Transcrip-tional regulation of a patatin-1 gene in potato. Plant MolBiol 14: 995–1006

Journet EP, Bligny R, Douce R (1986) Biochemical changesduring sucrose deprivation in higher plant cells. J BiolChem 261: 3193–3199

Kim S-R, Costa MA, An G (1991) Sugar response elementenhances wound response of potato proteinase inhibitorII in transgenic tobacco. Plant Mol Biol 17: 973–983

Koch KE (1996) Carbohydrate-modulated gene expressionin plants. Annu Rev Plant Physiol Plant Mol Biol 47:509–540

Koch KE, Nolte KD, Duke ER, McCarty DR, Avigne WT(1992) Sugar levels modulate differential expression ofmaize sucrose synthase genes. Plant Cell 4: 59–69

Koizuka N, Tanaka Y, Morohashi Y (1995) Expression ofa-amylase in response to wounding in mung bean.Planta 195: 530–534

Lei M, Wu R (1991) A novel glycine-rich cell wall proteingene in rice. Plant Mol Biol 16: 187–198

Lu C-A, Lim E-K, Yu S-M (1998) Sugar response sequencein the promoter of a rice a-amylase gene serves as atranscriptional enhancer. J Biol Chem 273: 10120–10131

Maas C, Schaal S, Werr W (1990) A feedback controlelement near the transcription start site of the maizeShrunken gene determines promoter activity. EMBO J 9:3447–3452

Macknight R, Lister C, Dean C (1998) Rice cDNA clonesOsGRP1 and OsGRP2 define two classes of glycine-richRNA binding proteins. Plant Physiol 117: 1527–1527

McElroy D, Rothenberg M, Wu R (1990) Structural char-acterization of a rice actin gene. Plant Mol Biol 14:163–171

Mikami K, Iwabuchi M (1993) Regulation of cell cycle-dependent gene expression. In DPS Verma, ed, Controlof Plant Gene Expression. CRC Press, Boca Raton, FL, pp51–68

Muller-Rober BT, Kobmann J, Hannah LC, Willmitzer L,Sonnewald U (1990) One of two different ADP-glucosepyrophosphorylase genes from potato responds stronglyto elevated levels of sucrose. Mol Gen Genet 224: 136–146

Murashige T, Skoog F (1962) A revised medium for rapidgrowth and bioassays with tobacco tissue cultures.Physiol Plant 15: 473–497

Ohta S, Hattori T, Morikami A, Nakamura K (1991) High-level expression of a sweet potato sporamin gene pro-moter: b-glucuronidase (GUS) fusion gene in the stemsof transgenic tobacco plants is conferred by multiple celltype-specific regulatory elements. Mol Gen Genet 225:369–378

Peto JV, Weisbeek PJ, Smeekens SCM (1999) Mannoseinhibits Arabidopsis germination via a hexokinase-mediated step. Plant Physiol 119: 1017–1023



Dow



ovember 2021

Purcell PC, Smith AM, Halford NG (1998) Antisense ex-pression of a sucrose non-fermenting-1-related proteinkinase sequence in potato results in decreased expressionof sucrose synthase in tubers and loss of sucrose-inducibility of sucrose synthase transcripts in leaves.Plant J 14: 195–202

Reynolds SJ, Smith SM (1995) The isocitrate lyase gene ofcucumber: isolation, characterization and expression incotyledons following seed germination. Plant Mol Biol27: 487–497

Roitsch T, Bittner M, Godt DE (1995) Induction of apoplas-tic invertase of Chenopodium rubrum by d-glucose and aglucose analog and tissue-specific expression suggest arole in sink-source regulation. Plant Physiol 108: 285–294

Ronne H (1995) Glucose repression in fungi. Trends Genet11: 12–17

Rose M, Albig W, Entian KD (1991) Glucose repression inSaccharomyces cerevisiae is directly associated with hexosephosphorylation by hexokinase PI and PII. Eur J Biochem199: 511–518

Sadka A, DeWald DB, May GD, Park WD, Mullet JE(1994) Phosphate modulates transcription of soybeanVspB and other sugar-inducible genes. Plant Cell 6:737–749

Salanoubat M, Belliard G (1989) The steady-state level ofpotato sucrose synthase mRNA is dependent on wound-ing, anaerobiosis and sucrose concentration. Gene 84:181–185

Sano Y, Sano R (1990) Variation of the intergenic spacerregion of ribosomal DNA in cultivated and wild ricespecies. Genome 33: 209–218

Sheen J (1990) Metabolic repression of transcription inhigher plants. Plant Cell 2: 1027–1038

Sheen J, Zhou L, Jang J-C (1999) Sugars as signaling mol-ecules. Curr Opin Plant Biol 2: 410–418

Sheu J-J, Jan S-P, Lee H-T, Yu S-M (1994) Control oftranscription and mRNA turnover as mechanisms ofmetabolic repression of a-amylase gene expression. PlantJ 5: 655–664

Sheu J-J, Yu T-S, Tong W-F, Yu S-M (1996) Carbohydratestarvation stimulates differential expression of ricea-amylase genes that is modulated through complicatedtranscriptional and posttranscriptional processes. J BiolChem 271: 26998–27004

Smeekens S (2000) Sugar-induced signal transduction inplants. Annu Rev Plant Physiol Plant Mol Biol 51: 49–81

Smeekens S, Rook F (1997) Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiol115: 7–13

Sturm A (1992) A wound-inducible glycine-rich proteinfrom Daucus carota with homology to single-strandednucleic acid-binding proteins. Plant Physiol 99:1689–1692

Sturm A, Chrispeels MJ (1990) cDNA cloning of carrotextracellular b-fructosidase and its expression in re-sponse to wounding and bacterial infection. Plant Cell 2:1107–1119

Uchimiya H, Kidou S-I, Shimazaki T, Aotsuka S, Taka-matsu S, Nishi R, Hashimoto H, Matsubayashi Y,Kidou N, Umeda M, Kato A (1992) Random sequencingof cDNA libraries reveals a variety of expressed genesin cultured cells of rice (Oryza sativa L.). Plant J 2:1005–1009

Viestra RD (1996) Proteolysis in plants: mechanisms andfunctions. Plant Mol Biol 32: 275–302

Walker DA, Sivak MN (1986) Photosynthesis and phos-phate: a cellular affair? Trends Biochem Sci 11: 176–179

Yu S-M (1999a) Cellular and genetic responses of plants tosugar starvation. Plant Physiol 121: 687–693

Yu S-M (1999b) Regulation of a-amylase gene expression.In K Shimamoto, ed, Molecular Biology of Rice, Chapter9. Springer-Verlag, Tokyo, pp 161–178

Yu S-M, Kuo Y-H, Sheu G, Sheu Y-J, Liu L-F (1991)Metabolic derepression of a-amylase gene expression insuspension-cultured cells of rice. J Biol Chem 266:21131–21137

Yu S-M, Lee Y-C, Fang S-C, Chan M-T, Hwa S-F, Liu L-F(1996) Sugars act as signal molecules and osmotica toregulate the expression of a-amylase genes and meta-bolic activities in germinating cereal grains. Plant MolBiol 30: 1277–1289

Yu S-M, Tzou W-S, Lo W-S, Kuo Y-H, Lee H-T, Wu R(1992) Regulation of a-amylase-encoding gene expres-sion in germinating seeds and cultured cells of rice. Gene122: 247–253

Zhou L, Jang J-C, Jones TL, Sheen J (1998) Glucose andethylene signal transduction crosstalk revealed by anArabidopsis glucose-insensitive mutant. Proc Natl AcadSci USA 95: 10294–10299

Ho et al.


Dow



ovember 2021

Documents

Sugar Coordinately and Differentially Regulates Growth- and