A Flooding-Lnduc D Xyloglucan €Do-Transglycosylase Homolog in Maize 1S Responsive to Ethylene and Associated Wit H Aerench Yma’

Plant Physiol. (7996) 7 72: 385-391

A Flooding-lnduc d Xyloglucan €do-Transglycosylase Homolog in Maize 1s Responsive to Ethylene and Associated wit h Aerench yma’

lmad N. Saab* and Martin M. Sachs Department of Crop Sciences, University of Illinois, Urbana, Illinois 61 801 (I.N.S., M.M.S.); and United States Department of Agriculture, Agricultura1 Research Service, Urbana, lllinois 61 801 (M.M.S.)

structural modifications leading to visible changes in cell Development of aerenchyma (soft cortical tissue with large in- and organ structure (Drew et al., 1979; Grineva et al., 1988). tercellular air spaces) in flooded plants results from cell-wall hy- Of these, the development of aerenchyma has been studied drolysis and eventual cell lysis and is promoted by endogenous extensively at the physiological leve1 (e.g. He et al., 1992, ethylene. Despite its adaptive significance, the molecular mecha- 1994; Brailsford et al., 1993). nisms behind aerenchyma development remain unknown. We re- Aerenchyma development results from the lysis of cells cently isolated a flooding-induced maize (Zea mays 1.) gene in cortical tissues and the ensuing formation of air spaces (wus/7005[gful;abbreviated as 7005) encoding a homolog of xyloglucan endo-transglycosylase (XET), a putative cell-wall-loosening and is proposed to involve a wide range of cell-degrading enzyme active during germination, expansion, and fruit softening. enzymes (He et al., 1994). The adaptive significance of XET and related enzymes may also be involved in cell-wall metab- aerenchyma lies in the fact that the tissue provides a low- olism during flooding-induced aerenchyma development. Under resistance pathway for air diffusion into submerged tissues flooding, 1005 mRNA accumulated in root and mesocotyl locations and, therefore, may promote flooding survival (Drew et al., that subsequently exhibited aerenchyma development and reached 1979). Despite its significance, however, the molecular maximum levels within 12 h of treatment. Aerenchyma develop- mechanisms behind the development of aerenchyma and ment was observed in the same locations by 48 h of treatment. other flooding-induced structural modifications are largely Treatment with the ethylene synthesis inhibitor (aminooxy)acetic unknown. It is established that aerenchyma development acid (AOA), which prevented cortical air space formation under flooding, almost completely inhibited 1005 mRNA accumulation in in flooded tissues is associated with increased activity of both organs. AOA treatment had little effect on the accumulation of cellulase, and that both are promoted by the accumulation mRNA encoded by adhl, indicating that it did not cause general of ethylene (Drew et al., 1979; He et al., 1994). Thus, it is suppression of flooding-responsive genes. Additionally, ethylene likely that genes involved in cell-wall loosening or hydro- treatment under aerobic conditions resulted in aerenchyma devel- lysis under flooding are induced in response to ethylene. opment as well as induction of 7005 in both organs. These results Besides the observed increase in cellulase activity, how- indicate that 7005 is responsive to ethylene. Treatment with anoxia, ever, there is no available information about the enzymes which suppresses ethylene accumulation and aerenchyma develop- involved in the development of aerenchyma or other flood- ment, also resulted in 7005 induction. However, in contrast to flooding, AOA treatment under anoxia did not affect 1005 mRNA ing-induced structural changes. accumulation, indicating that 7005 is induced via different mecha- We recently cloned a flooding-induced gene from maize nisms under flooding (hypoxia) and anoxia. (Zea mays L.), which encodes a homolog of XET, a putative cell-wall-loosening enzyme (gene wus12005(gfu), Peschke and Sachs, 1994; Saab and Sachs, 1995; referred to as gene 2005). The induction of 1005 mRNA was specific to oxygen Plants respond to flooding by undergoing changes at the deprivation among a range of stresses, including heat molecular, biochemical, and cell structural levels. To date, shock, salt, and cold (Peschke and Sachs, 1994). 1005, which the majority of molecular-leve1 research on flooding re- is one of the first flooding-responsive genes that does not sponses has focused on the enzymology of energy produc- encode an enzyme of Glc-P metabolism, is proposed to be tion, namely the induction of genes encoding enzymes of associated with the onset of structural modifications in- Glc-P metabolism (egADH, enolase, and glyceraldehyde- 3-P dehydrogenase). These enzymes allow limited energy duced by flooding (Saab and Sachs, 1995). production in the face of limited oxygen supply and thus Xyloglucans, the substrates for XET, are composed of aid in survival during short-term flooding (reviewed by /3-1,4-linked glucans with xylosyl side chains and can form Sachs et al., 1996). Flooded plants also undergo significant strong hydrogen bonds with cellulose microfibrils (Valent and Albersheim, 1974). As such, xyloglucans are proposed to act as tethers that cross-link cellulose microfibrils and Supported by U.S. Department of Agriculture/National Re- contribute to cell-wall strength (Fry, 1989; Passioura and search Initiative Competitive Grants Program grant no. 95-37100- 1563 to M.M.S. and I.N.S. * Corresponding author; e-mail [email protected]; fax 1-217-333- Abbreviations: ADH, alcohol dehydrogenase; AOA, (ami- 6064. nooxy)acetic acid; XET, xyloglucan endo-transglycosylase. 385 386 Saab and Sachs Plant Physiol. Vol. 112, 1996

Fry, 1992). The primary activity of XET is transglycosyla- Tissue Sampling and Histology tion, the cutting and rejoining of xyloglucan chains. How- Ten seedlings were harvested after 48, 72, or 96 h of ever, depending on substrate conditions, some XETs may flooding treatment, and primary roots and mesocotyls also cause hydrolysis of xyloglucans (Fanutti et a]., 1993; were examined for structural modifications as described S.C. Fry, personal communication). Consistent with its pro- below. Locations in each organ exhibiting the first signs of posed role as a cell-wall-loosening enzyme, increased modifications (visible cell deformation, lysis, or air space activity of XET was associated with severa1 cell-wall- formation) were selected for RNA extraction in subsequent loosening and hydrolysis processes, including expansion experiments. In the primary root, a 2-cm location starting at (Hetherington and Fry, 1993), fruit softening (Redgwell 2 cm below the kernel was selected for analysis. In the and Fry, 1993; Maclachlan and Brady, 1994), and responses mesocotyl, a 2-cm location starting at 3 cm below the to water deficit (Wu et al., 1994). coleoptile was selected for analysis. These locations were The hypothesis behind this work is that gene 2005 encodes a flooding-induced XET or related enzyme that is used in a11 experiments. involved in cell-wall metabolism associated with develop- For structural analysis, primary roots and mesocotyls ment of aerenchyma. Our objectives in this study are first, were separated from seedlings and sectioned by free-hand at 1-cm intervals along each organ. Sections were stained to characterize this gene and its regulation by flooding and with 1%toluidine blue, rinsed with water, and mounted on anoxia; second, to test its association with flooding- microscope slides. Sections were examined immediately induced air space formation leading to aerenchyma devel- under a microscope with 63 magnification. Photographs opment; and third, to determine if it is responsive to eth- x were taken using Kodak T 160 film. ylene, a promoter of aerenchyma development.

MATERIALS AND METHODS RNA Extraction and Northern Analysis Experimental Conditions Total RNA was extracted from 20 root or mesocotyl sections as described by Saab et al. (1995), and 30-pg sam- A11 experiments were conducted on the maize (Zea mays ples were fractionated on 1%formaldehyde gels. RNA was L.) inbred line B73Ht. Kernels were germinated for 4 d at capillary transferred to nylon membranes (MagnaGraph, 28°C in the dark, and 25 seedlings of uniform average Micron Separations, Westborough, MA)' using 10X SSC length with preemergent shoots (leaves remaining within (1.5 M NaCl, 0.15 M trisodium citrate) for 20 h. RNA was the coleoptile) were selected. For flooding treatments (hyp- cross-linked to membranes by exposure to UV light for 1 oxia), seedlings were placed in 2-L containers containing min (UV Stratalinker, Stratagene). Northern hybridizations 1.2 L of flooding buffer (5 mM Tris-C1, pH 8, 100 mg L-' were performed under high-stringency conditions as fol- ampicillin). Seedlings were maintained in a vertical orien- lows: blots were prehybridized in hybridization solution tation and submerged in flooding buffer so that coleoptile for 3 h at 42°C followed by hybridization in fresh solution tips were just below the liquid surface. Uncovered contain- (plus probe) for 20 h at 42°C. Hybridization solution com- ers were incubated at 28°C in an aerated chamber for the position was 50% formamide, 6X SSC, 20 mM phosphate number of hours indicated. For anoxia treatments, seed- buffer, pH 6.8, 1%SDS, 1X Denhardt's solution, 5% (w/.v) lings were submerged in flooding buffer and sealed in a dextran sulfate, and 100 mg/mL sheared salmon sperm 2-L glass container. A plastic tube connected to an argon DNA. After probing, blots were washed with 2X SSC, 0.2% gas tank was sealed into the screw cap, and the container SDS at room temperature for 15 min, then at 65°C for 30 was overfilled with buffer before sealing to remove as min, and finally with 1X SSC, 0.1% SDS at 65°C for 20 min. much air as possible. Seedlings were bubbled continuously Solutions were prewarmed to the appropriate tempera- with argon gas, which was vented into a water-filled bea- tures before washing. Blots were stripped by soaking in ker (procedures similar to those of Sachs et al., 1980). The 0.2X SSC, 0.1% SDS at 100°C for 15 min. container was incubated at 28°C for the duration of the experiments. For inhibitor treatments, AOA was added to the germination medium and flooding buffer at a concen- Cloning, Sequence Analysis, and Probes tration of 0.7 mM. Other experimental conditions were the Details of cloning of genomic and full-length cDNA se- same as those used for flooding and anoxia treatments. quences representing 2005 were reported by Saab and For experiments with exogenous ethylene, 25 seedlings Sachs (1995). Homologous sequences in the GenBank were (3 d old) were placed on moist germination paper, which identified using a BLAST search (National Center for Bio- was then rolled loosely. The rolled paper (30 cm high) was technology Information, Bethesda, MD). Amino acid se- placed in 1 cm of water in a 2-L glass container fitted with quences were aligned with the aid of MacDNASIS rro a cap connected to a tank containing 100 pL L-' ethylene in sofhvare (Hitachi Software Engineering Co., San Bruno, air. The seedlings, including root tips, remained above the CA). For northern analysis, blots were probed with a 0.8-kb water leve1 for the duration of the experiments to avoid potential hypoxic conditions. Ethylene gas was bubbled in Names are necessary to report factually on available data; the water at the bottom of the container to maintain high however, the U.S. Department of Agriculture (USDA) neither humidity and was vented into a water-filled beaker to guarantees nor warrants the standard of the product, and the use monitor gas flow. AI1 experiments in this study were re- of the name by the USDA implies no approval of the product to the peated in their entirety one or two times. exclusion of others that may also be suitable. A Flooding-lnduced Xyloglucan Endo-Transglycosylase Homolog from Maize 387

10 20 30 40 50 partial-length 1005 cDNA (pZmL1005, Peschke and Sachs, 50 1994) or with a probe containing the 3'UTR of the clone 50 Soybean 1 50 generated using an interna1 SstI site in the clone (Fig. 1)and Arabidopsis 50 an XhoI site in the vector. The latter produced a single band 100 100 on Southern analysis of genomic DNA, suggesting that it is 100 specific to only one gene (Peschke and Sachs, 1994). The 100 other probes used were ADHl cDNA (clone pZmL793), 150 150 which served to distinguish treatment effects on 1005 from soybean 101 150 effects on other flooding-responsive genes, and clone Arabidopsis 101 150 pZmL1055, an unidentified clone the transcript leve1 of 200 200 which is unaffected by oxygen deprivation (Sachs, 1994) 200 and, as such, served as a loading control. Even loading was 200 also verified by examining ethidium bromide-stained ribo- 250 250 soma1 bands in the gels and on the membranes. Probes 250 were labeled with [(U~~PJ~ATPor [cy3*P]dCTIJ (3000 Ci/ Arabidopsis 201 250 mmol, ICN) by random priming (Prime-It 11, Stratagene). 300 300 300 300 RESULTS Figure 2. The comparison of the deduced amino acid sequence of 1005 and XET sequences from wheat (Okazawa et al., 1993), soy- 7005 1s Homologous to XET bean (Zurek and Clouse, 1994), and Arabidopsis (Xu et al., 1995) in the GenBank. Dark regions denote identities among two or more of Figure 1 shows the transcriptional unit of 1005 and an the sequences. additionall20 bp 5' to the ATG start site. Comparison with the full-length cDNA revealed that this gene contains a single intron, 125 bp long, starting with GT at the 5' end Evidence for the identity of the deduced 1005 protein and ending with AG at the 3' end (the intron is denoted by comes from comparison with XET sequences in the data- lowercase letters). At least one XET encoding gene, TCH4 bases, which shows that 1005 is homologous to a11 known from Arabidopsis thaliana, was also found to include only XET sequences. Figure 2 shows a comparison of 1005 with one intron (Xu et al., 1995). It is interesting that the intron three XET sequences isolated from vegetative tissues. The position relative to the translation initiation site in 1005 and wheat sequence (Okazawa et al., 1993) is the only known TCH4 differs by only 3 bp, indicating an evolutionary XET homolog from a monocot species in addition to 1005. relationship between the two genes. Furthermore, recombinant proteins produced from the soybean (BRU1, Zurek and Clouse, 1994) and Arabidopsis (TCH4, Xu et al., 1995) sequences were found to possess 1 CGGCACGAGA TCGCACGCAC CATCGTCCTG CCGATCGATC GCAGCCAGCC AGTCTTGCTG XET activity in vitro (Xu et al., 1995; S. Clouse, personal 61 AGAGACGAAC TGAAGCCTGA AGTGAACCGA TCGATCGATC GATCGAGCGA GCGAGCTCCC communication). Both of these sequences are inducible by 121 mAAGGCGT TGATTCTGGC GGCGGTGCTG CTGCTGCAGC ACGGGGGAGC CGCGAGCGCC treatment with a brassinosteroid growth regulator, and 181 GGCGGCAACT TCTACCAGGA CGTGGACATC ACCTGGGGCG ACGGCCGCGG CAAGATCCTC TCH4 is additionally inducible by heat shock and mechan- 241 GACAACGGCC AGCTCCTGAC GCTGTCCATG GACAGGTCCT CCGGCTCGGG CTTCCAGTCC ical stimulation (Xu et al., 1995). 301 AAGGCCCAGT ACCTCTACGG CCGCTTCGAC ATGCAGCTCA AGCTCGTCCC GGGGGACTCC 361 GCCGGCACCG TCGCCACC'M CTATGTACgt actactacgt acatactaca ccgccgccgc A11 known XET homologs, including 1005, share an iden- 421 ctcctcgtcc tagcttcgta tatsttcgct gtcgtctctc tctctcgcgc gctgaacaca tical or near-identical 11-amino acid stretch (Fig. 2, under- 481 tgtcgtcggt cacgcacgca cgcgcacagc TTTCGTCGCA GGGTTCGCAG CACGACGAGA lined), which is proposed to contain the active site of 541 TCGACTTCGA GTTCCTGGGG AACGCGAGCG GGGAGCCGTA CACGGTGCAC ACGAACGTGT p-glucanases from Bacillus subtilis (Borriss et al., 1990). As 601 ACAGCCAGGG GAAGGGCGGG CGGGAGCAGC AGTTCCGGAT GTGGTTCGAC CCCACGGCGG is expected for proteins that are putatively targeted to cross 661 CCTTCCACGC CTACTCCGTG CTGTGGAACC CCGCCCACGT CGTCTTCTAC GTGGACGGCG the plasma membrane, 1005 contains a hydrophobic se- 721 TCCCCATCCG GGAGTTCCGG CGCCGCGGCG ACGGGACCGT GCCGTTCCCG ACGTCGCAGC quence at the N terminus (first 20 residues). Similar se- 781 CGATGCGGGT GTACGCCAGC GTGTGGCACG CGGAGGAGTG GGCCACGCAG GGGGGGCGCG quences have been observed in other XET homologs (e.g. 841 TCCGGACCGA CTGGTCCAAG GCGCCCTTCG TCGCGTCGTA CCGCGGGTAC GCCGCCGCCG Zurek and Clouse, 1994; Arrowsmith and de Silva, 1995; Xu 901 GGTGCACCGC GCCGGACGCC GCCGCCTGCG CGCGCTCCAA CGGCGCATGG ATGTCGCAGG et al., 1995). 961 mGACAG CGCCGGCCAG GAGCAGCTCC GCCGGGCGCA GGCCAGCTAC ATGATCTACA 1021 ACTACTGCAC CGATAAGTAC CGGTTCCCGC AGGGCCCGCC GCCCGAGTGC TCGTCGCCGG 1081 CCAAGTAGTA GATGAGTAGA ATTGATCGGA TACAGAGAGC AATCAATTAA TTAAATCGAC lnduction of 7005 and Aerenchyma by Flooding 1141 CCGTCGCTTG GTTTTTGGTT TACACATTGT ACTACGGCAT AAGTTCGGCG GATATATGTA 1201 TACGTAGACG GCTAGACGCC ATGCATGTGT CTTGGCATGT ATCGTATTCG TATATATCGT To begin to address the association of 2005 expression 1261 AATTCGTATG TACCTCAGTA TACCTGCATC GTACA'X'TAC ATGCATATAT ATCTCCCTAT with flooding-induced structural modifications, we first 1321 ATATAAATAT ATATGGTMT ATTA examined the effects of the flooding conditions used in this study on root and mesocotyl structure. Cortical air spaces Figure 1. The nucleotide sequence of 1005. Predicted translation initiation site and an Sstl restriction site used to generate a 3' end were generally detected by 48 h of flooding in both organs, gene-specific probe are underlined. The intron location, determined and aerenchyma development was clearly discernible in by comparison with the complete cDNA, is denoted in lowercase the root (Fig. 3) and the mesocotyl (data not shown) by letters. 72 h. In the primary root, air space formation was first 388 Saab and Sachs Plant Physiol. Vol. 112, 1996

tion treatee w , d seedlings with AOA inhibiton a , f etho r - ylene synthesis (Yang and Hoffman, 1984), and examined effects it aerenchymn o s a developmen accumulatiod an t n of 1005 mRNA under flooding controla s A . e als- w ,o ex amined AOA effects on ADH1 mRNA accumulation to determin actA s AO indiscriminatel f ei genn yo e expression during flooding a necessar. This wa s y control because, depending on concentration, AOA can act as a general inhibito enzymef o r s requiring pyridoxal phosphate, lead- potentiallo t g in y toxic effects (Imaseki, 1991). Preliminary experiments were conducte determino dt appropriate eth e AOA concentration for this study. Seedlings were treated with 0.1, 0.3, or 0.7 mM AOA and examined for cortical air space formation under flooding. Results indicated that moss wa t M effectivm 7 0. reducin n t i ea A AO g aerenchyma development seedling0 3 f o t s Ou . examined, none exhibited air space formation in the mesocotyl and 4 showed reduce r spacdai e formatio rooe floodf th o t n i afteh 8 -4 r ing. Thus, this concentration was chosen for analysis of mRNA accumulation. Treatmen t almosA witAO h t completely abolished 1005 mRNA accumulation floodinwithif o h 4 n2 roogn i t mesocotyd an l tissues compared with untreated controls othee th n r (FigO hand . accumulatioe 5) . th , f ADHno 1 transcripts was affected to a much lesser extent by this treatment (Fig Thi. .5) s indicate treatmenA d AO tha e th t t did not cause a general inhibition of flooding-induced gene expression t specificallbu , y affected 1005 mRNA accumulation maizn I . e seedling similaf o s t a r A ageAO , 0.7 mM was shown to inhibit ethylene accumulation in AerenchymFigur) (A . e3 a inductio primare th n i y roo floodingy b t . roots under water deficit. Also, its effect on root elonga- Seedlings were harvested at 0 (A) and 72 h (B) after flooding, and sections were taken at 3 cm below the kernel. Sampling location was tion was similar to that obtained with silver thiosulfate, determined from result f preliminaro s y experiments that identified an inhibitor of ethylene action, suggesting that AOA at locations exhibiting early signs of aerenchyma development, as de- this concentration affected mainly ethylene synthesis scribe "Materialn di Methods.d an s " (Spolle Sharpd nan , 1994 . SpollenW ; , personal communication). These results suggest that 1005 mRNA accu- observed withi 2-cna m location startin t approximatelga y belom 2 c kernele wth , wherea mesocotye th n si spacr ai l e ROOT MESOCOTYL formation was first observed within a 2-cm location start- t approximatela g in belom c coleoptilae y3 w th r nodey B . 1005 96 h of flooding, air spaces were prevalent in all locations •'HI of both organ e primarth f s o e excepapica yth m c r 2 lfo t root. Similarly, Drew et al. (1979) reported no aerenchyma apican i l region f floodeso d nodal roots. ADH1 The accumulation of 1005 mRNA was examined in the WWW ~Wf w above-mentioned locations exhibiting early signs of aeren- ••••• .1 chyma development. Transcript level was barely detectable eithen i r organ under aerobic conditions. Under flooding, 1055 •Ml •••• however, 1005 mRNA accumulate botn di h organ- ad n i s vance of cortical air space formation and reacheh 36 dh a 24 maxi h 12 - h O h 36 h 24 h O12 h f treatmeno withim h mu 2 n1 t (Fig . Transcrip.4) t levels remained high through 144 h of flooding (data not shown). inductioe FigurTh . e4 f 100 nprimaro ADHe d th 5 an n i 1 y rood an t mesocotyl by flooding. Total RNA was extracted from locations exhibiting early aerenchyma development in the primary root and Is 7005 Inductio Floodiny nb gResponsa o t e mesocotyl (Fig. 3). A 2-cm location starting at 2 cm below the kernel Endogenous Ethylene? was selecte primare th n di y 2-c a root d m ,an location startinm c 3 t ga below the coleoptilar node was selected in the mesocotyl. RNA was Since aerenchyma developmen promotes i t endogey db - fractionated and blotted, and blots were hybridized as described in nous ethylene (Dre t wal.e ,t al.e 1979 ,e 1992)H ; e th , "Material Methods.d an s " Blots were hybridized with pZmUOOS question arises whether endogenous ethylene is a trigger (1005 partial cDNA), stripped thed an n, hybridized with pZml_793 for 2005 induction under flooding addreso T . s this ques- (ADH1 cDNA) and pZmL1055 (loading control). A Flooding-lnduced Xyloglucan fndo-Transglycosylase Homolog from Maize 389

ROOT MESOCOTYL treatmen n 200o t 5 mRNA accumulation under flooding (hypoxia) versus anoxia (Fig , usin.7) g200a 5 gene-specific prob confiro et m tha same tth e gen detectes ei d under both 1005 if 1 conditions. Results showed that 1005 was strongly induced f anoxio h roon d mesocotyb4 ai y 2 an t l tissues despite treatment with AOA. Under flooding (hypoxic) conditions, however, AOA treatment almost completely abolished ADH1 • f 2005 induction in both organs (Fig. 5). This points to differential mechanism regulatioe th r sfo 2005f no , depending on the severity of oxygen deprivation; the gene appears to I 105I 5 •• inducee b ethyleny b d e accumulation under hypoxid aan by an alternative mechanism under anoxia.

orC T ,&o DISCUSSION Figure 5. The effect of AOA treatment on induction of 1005 and Althoug e majoritth h f knowyo n flooding-responsive primare ADHth n i 1 y f floodingmesocotyrooo d h an t A 4 2 AO y . b l genes encode Glc-P metabolism enzymes, it was not sur- germinatioaddee s th wa o dt M floodind m an n7 a0. t g media- un ; prising tha a flooding-responsivt e gene encodin- ho a g treated controls were germinated and flooded under identical conditions. Tissue samplin experimentad gan l procedures wer same eth e molog of XET, the putative cell-wall-loosening enzyme, as those describe legene th n f di Figurdo . e4 woul isolatede db . After all, flooded tissues undergo sub- stantial structural modifications, manifested mainly in the developmen f aerenchymao t s plausibli t i d an e, that this mulation under floodin responsivs gi ethyleno t e e accu- would involv e activatioth e f geneo n s encoding wall- mulation, and thus, induction of 2005 and aerenchyma loosenin degradind gan g enzymes, including XET. development appear to share a common trigger. Also, XET has generated considerable interest among investi- resulte th s suggest that inductio ADHf no floodiny 1b s gi gator walf so l properties during growt developmend han t not responsive to ethylene accumulation. and responses to environmental stress. Until fairly recently, cell-wall mechanical properties pertainin walo t g l 1005 Is Induced by Ethylene Treatment under loosening and cell expansion were described mainly using Aerobic Conditions mathematical models (reviewe Passiouray db , 1994). How- To further test the responsiveness of 2005 to ethylene, we ever identificatioe th , putativd an T eXE f wall-looseninno g treated seedlings with exogenous ethylene under fully aer- factors such as expansins (McQueen-Mason et al., 1992) has obic conditions to determine whether the gene can be offere a biochemicad l explanation, albeit partial f walo , l inducee hormonth y b d e without flooding. mRNA accu- loosening and its interactions with overall wall metabo- mulatio s examinenwa same th en di location e prith -n si lism. The activity of XET has been observed in many spe- mary root and the mesocotyl that were used in the flooding d ciefoune correlateb an s o t d d wit a hwid e rangf o e anoxid an a experiments, usin g200a 5 gene-specific probe processes, including expansive growth (Fry et al., 1992), (Fig . Thi6) . s confirmed thaethylene-responsive th t e gene wall adjustment responsn i s wateo t e rt al. e defici ,u (W t e flooding-responsive samth i th ss a e e gene. Treatment 1994), and fruit ripening and softening (Redgwell and Fry, with ethylene for 48 h resulted in the accumulation of 1005 1993; Maclachlan and Brady, 1994). These observations mRN botAn i h organs additionn (FigI . 6) . , this correlated with the induction of aerenchyma in both organs (data not ROOT MESOCOTYL shown). On the other hand, ethylene treatment did not cause the accumulation of ADH1 mRNA in either organ (Fig. 6). This confirmed that the seedlings did not experi- 1005 ,1 ence hypoxia during the treatment, since adhl is induced eve mily nb d hypoxia (Pau Ferld lan , 1991) resulte .Th s also confirmed that, unlike 2005 t responsiv, no adhl s i o t e ADH1 ethylene. Differential Regulatio f n1005o Floodiny b Anoxid gan a 1055 *• In an earlier study, 2005 was induced in maize seedlings Oh 48h Oh 48h that were sealed in drowning buffer, a condition of near anoxia (Peschk Sachsd ean , 1994). Under anoxic conditions, inductioe FigurTh . e6 f 100no ethyleny 5b e under aerobic conditions. Seedlings were treated wit0 /j.L10 h L~' s ethylena r ai n i e however, ethylene accumulation is inhibited along with describe "Materialn di Methods.d an s " Tissue samplin experid gan - aerenchyma development (Dre t wal.e , 1979; Brailsfort de mental procedures were the same as those described in the legend of al., 1993). This suggested thae inductioth t f 200no y 5b Figure 4. Blots were hybridized with a 3' end 7005-specific probe to anoxia occurabsence th n si ethylen f eo e accumulationo T . confirm thaethylene-responsive th t e on e e e samgenth th s s a ei e investigate this possibility comparee w , A effece AO dth f o t induced by hypoxia. 390 Saa d Sachban s Plant Physiol. Vol. 112, 1996

ROOT MESOCOTYL ity assay vitrn i s o were founpredominantle b o dt y trans- glycosylases, at least one possessed significant hydrolytic activity (Fanutti et al., 1993). 1005 may contribute to wall 4 • 100M 5 *» | metabolism directl hydrolyziny yb g xyloglucan r otheso r matrix polysaccharides. Alternatively a transglycosy s a , - lase, 100contributy 5ma walo et l degradation indirectly yb ADH1 M• • facilitating accesdense th o est wall matrix regulatiny b r ,o g ;fet the accumulation of biologically active oligosaccharides generate hydrolasesy db assessmenn A . enzymatie th f o t c 1055 II activit recombinane th f yo t protei s currentlni y underway to address these possibilities. e 100Th 5 protei alsy involvee ob nma earln i d y events leading to structural modifications. Indeed, XET encoding TCH4 from Arabidopsis is rapidly induced by touch and is treatmenA effece FigurAO Th f o n 100. t o 7 et 5 induction under propose e involveb o t d earln i d y respons o changint e g floodin d anoxiaan g . AOA-treated seedlings were submergen i d environments (Xu et al., 1995). Clearly, a role for the 1005 flooding buffe r treateo r d anaerobicall describes a y "Materialn i d s protein in the development of aerenchyma or other struc- Methods.d an " Tissue samplin experimentad gan l procedures were same th thoss ea e describe legene th n f i dFigurdo . Blote4 s were tural modifications would have to be in concert with an hybridized with a 3' end /005-specific probe to confirm that the arra hydrolytif yo c enzyme brino st g abou changa t thif eo s anoxia-induce e induceon d same e gen y hypoxiath th d b s s ei a . magnitude. Thi alss si o supporte observatioe th y db n that 1005 mRNA accumulation under anoxia is not sufficient for aerenchyma development, although actual protein levels indicate that XET activity is associated with a broad range have not been determined. In addition, the fact that aeren- of physiological events from seed germinatio fruio nt t rip- chyma develops only in the inner cortex suggests that cells ening, probably throug actioe hth multiplf no e forme th f so in that regio specifie nar c target r ethylensfo e action (Bal- enzyme maizn I . e cell walls, however, xyloglucan content usk al.t ae , 1993 thad an )t genes involve thin di s procese sar is relativel (Carpitw ylo Gibeautd aan , 1993), which raises regulated in a coordinated and tissue-specific manner, questions about the importance of XET and other xyloglu- leading to a form of programmed cell death. canase controllinn si g wall metabolis thimn i s species. Nev- s interestini It g that 1005 mRNA accumulated under ertheless. (1994al t e )u founW , d activit T thaXE t growyn i - anoxia despite treatment with AOA, even though the same ing tissues of maize was higher than that in several dicot treatment almost completely abolished mRNA accumula- species, despite the low abundance of xyloglucans in maize tissues. Also, level f 100so 5 mRNA were highly abundant tion under hypoxia (flooding). This raises a question about under flooding (Fig , suggestin4) . d relategan T thad XE t possible differential mechanism f 200o s 5 induction under enzyme play ma ysignificansa t rol cell-waln ei l metabo- flooding versus anoxia, and suggests that the gene may lis floodemn i d maize tissues. play different roles under anoxia and hypoxia. In maize e moleculath n O r levelsequenceT XE , s have been iso- roots, anoxi s beeaha n show suppreso t n s ethylene accu- lated from several species, and in some species more than mulation as well as aerenchyma development (Drew et al., cDNT s beeXE A ha e n on identified (e.g. Okazaw t al.ae , 1979) t measur. Althougno d di e e ethylenhw e accumula- 1993; Arrowsmit Silvae d d , han 1995) . Overall currene th , t tion under our anoxia conditions, we observed a lack of molecula genT re picturXE expressioe th f eo n indicates that cortica r spacai l e formatio roon mesocotyd i an t l tissues. the enzyme is encoded by multiple genes (Peschke and This indicates that 2005 induction by anoxia occurred de- Sachs, 1994; Arrowsmith and de Silva, 1995; Xu et al., 1995) spite an apparent inhibition of ethylene accumulation (or thad an t some gene inducee ar s responsn i d growto et h t affecteno s AOAy b d wa explaiy t i action) . ma y d nwh an , regulators (Zure d Clousekan , t al.1994e ,u 1995X ; ; this This result also suggests tha t leasseparata o t tw t e mecha- work) and/or environmental stimuli (Xu et al., 1995; this nisms exis r 100fo t 5 induction mechanisa : m tha activs i t e work). under hypoxia or flooding, which requires ethylene accu- associatioe Th 200f no 5 with flooding-induced structural mulation, and an additional or alternative mechanism ac- modifications is indicated by the observation that the gene tive during anoxia independent of ethylene accumulation. is induced in regions of the primary root and mesocotyl Althoug develot h theno o yd p localized aerenchym, se r ape that exhibit early signs of aerenchyma development under anaerobically treated seedlings develop regions of tissue flooding associatioe Th . alss ni o supporte apparene th y db t softening in the primary root and mesocotyl, which even- responsiveness of 2005 induction under flooding to endog- tually spread to the entire organ. This may involve the enous ethylene, a promoter of aerenchyma development, action of 2005. It is possible that, in the absence of ethylene s responsivenesit y b d an exogenouo t s s ethylene under production during anoxia e targetinth , f programmego d aerobic conditions, which also promotes aerenchyma cell death specifically to the cortex and the ensuing aeren- development. chyma developmen loste ar t. Another possibilit thats yi n i , 100e th role 5f eTh o protei floodinn ni g responsey ma s contrast to 2005, other genes associated with aerenchyma depend on whether it is involved in transglycosylation or development are suppressed due to the lack of ethylene hydrolysi vivon i s . Although most XETs subjecte activo dt - accumulation during anoxia. Alternatively e accumuth , - A Flooding-lnduced Xyloglucan Endo-Transglycosylase Homolog from Maize 391 lated 1005 mRNA may simply not be translated under ethylene in nitrogen- or phosphate-starved roots of Zea mays L. anoxia. To begin to address these questions, the activity of during aerenchyma formation. Plant Physiol 98: 137-142 XET and its responsiveness to ethylene should be exam- Hetherington PR, Fry SC (1993) Xyloglucan endotransglycosylase activity in carrot cell suspension during cell elongation and ined under hypoxic and anoxic conditions. These studies somatic embryogenesis. Plant Physiol 103: 987-992 and other investigations on the role of 2005 in flooding Imaseki H (1991) The biochemistry of ethylene biosynthesis. In AK responses are currently underway. Mattoo, JC Suttle, eds, The Plant Hormone Ethylene. CRC Press, Boca Raton, FL, pp 1-20 ACKNOWLEDCMENTS Maclachlan G, Brady C (1994) Endo-1,4-P-glucanase, xyloglu- canase, and xyloglucan endo-transglycosylase activities versus We are grateful to Dr. John Cheeseman (Department of Plant potential substrates in ripening tomatoes. Plant Physiol 105: Biology, University of Illinois at Urbana-Champaign) for valuable 965-974 assistance in structural analysis and for the use of equipment in his McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two endogenous proteins that induce cell wall extension in plants. laboratory. We are also grateful to Dr. William Spollen (University Plant Cell4: 1425-1433 of Missouri-Columbia) for his helpful input in the ethylene exper- Okazawa K, Sato Y, Nakagawa T, Asada K, Kato I, Tomita E, iments and to Dr. Subbaiah Chalivendra in our laboratory for Nishitani K (1993) Molecular cloning and cDNA sequencing of helpful suggestions. endoxyloglucan transferase, a nove1 class of glycotransferase that mediates molecular grafting between matrix polysacchar- Received April 3, 1996; accepted June 6, 1996. ides in plant cell walls. J Biol Chem 268: 25364-25368 Copyright Clearance Center: 0032-0889 / 96 / 112/ 0385 / 07. Passioura JB (1994) The physical chemistry of the primary cell The GenBank accession numbers for the sequences described in wall: implications for the control of expansion rate. J Exp Bot 45: this article are U15964 (gene) and U15781 (cDNA). 1675-1682 Passioura JB, Fry SC (1992) Turgor and cell expansion: beyond the Lockhart equation. Aust J Plant Physiol 19: 565-576 LITERATURE ClTED Paul AL, Ferl RJ (1991) Adhl and Adh2 regulation. Maydica 36: 129-134 Arrowsmith DA, de Silva J (1995) Characterization of two tomato Peschke VM, Sachs MM (1994) Characterization and expression fruit expressed cDNAs encoding xyloglucan endo-transglycosylase. of anaerobically induced maize transcripts. 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