Planta (1988)175:115-120

P l ~ J n ~ 9 Springer-Verlag 1988

Hormonal regulation of gene expression in the "slender" mutant of barley (Hordeum vulgate L.) Peter M. Chandler CSIRO Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia

Abstract. The "slender" mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA3). Expression of GA3-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants. Production of o~-amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA3, and was prevented by ABA. In contrast, a-amylase was produced by half-grains of the slender mutant in the absence of added GA3, although it was still reduced by ABA. The spectrum of o~-amylase mRNAs in "slender" embryoless half-grains incubated in the absence of added GA3 was the same as in WT endosperm half-grains incubated in the presence of GA3. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA3. In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on a-amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA. Key words: Abscisic acid and gene expression Aleurone - 0~-Amylase - Gibberettin and gene expression - Hordeum (GA mutant) - Mutant (barley, GA).

Introduction Mutants of higher plants with alterations in their level of, or response to, individual hormones have Abbreviations: ABA = abscisic acid; AMV = avian myeloblasto-

sis virus; GA=gibberellin; GA1 = gibberellin A1; GA3 =gibberellic acid; WT = wild-type

played an important role in understanding the involvement of hormones in particular physiological processes. Notable examples are the dwarf mutants of pea and corn (gibberellin A1 [GA1] involvement in internode elongation: Ingram et al. 1984; Spray et al. 1984), viviparous mutants of corn (abscisic acid [ABA] involvement in the prevention of premature germination: Moore and Smith 1985; Neill et al. 1986), and "wilty" mutants of tomato (ABA involvement in stomatal regulation: Tal 1966). The "slender" mutant of barley isolated by Foster (1977) exhibits properties associated with treatment of plants with high concentrations of GA (long, narrow leaves, long internodes), and phenotypically resembles the wild-type (WT) plant treated with GA. However the biochemical basis for this phenotype is not known (see Discussion). The aleurone of barley caryopses responds to applied gibberellic acid (GA3) by synthesis and secretion of 0~-amylase. In the absence of GA3, or in the presence of both GA3 and ABA, very little a-amylase is produced. These effects of applied hormones have been found to operate via regulation of c~-amylase mRNA levels, in part at least by regulating the transcription rates of 0~-amylase genes (Jacobsen and Beach 1985; Zwar and Hooley 1986). In this paper the response of the "slender" mutant to applied GA3 and ABA at the biochemical level is described, principally in terms of the wellcharacterized aleurone response to these hormones. We have used hybridization of appropriate nucleic acids - either copy DNA (cDNA) clones or oligonucleotides derived from the sequences of cDNA clones - to monitor expression of particular mRNAs whose levels are regulated by GA3 or ABA application (Thomas 1980). This allows us to investigate certain aspects of hormone responses in greater detail than currently provided by other assays.

116

P.M. Chandler: Hormones and gene expression in slender barley

Material and methods Hormones. Gibberellic acid and abscisic acid ( ( •

cis-trans

isomer, 99 + %) were purchased from Sigma Chemical Co., St. Louis, Mo., USA and used at I ~tM and 25 gM, respectively.

Plant material. Seed stocks of Hordeum vulgare L. cv. Herta, which were either WT or segregating for the slender mutation (seed derived from heterozygous plants), were obtained from C.A. Foster, Welsh Plant Breeding Station, Aberystwyth, UK, and grown in a glasshouse: these lines are designated CPI107276 (segregating for "slender") and CPI107277 (nonsegregating). "Slender" plants do not set seed so a pure seed stock is unavailable. Heterozygous plants cannot be distinguished from homozygous WT plants, so heads were harvested from individual plants of CPI107276 and tested for heterozygosity as described below. Seed from heterozygous plants was then pooled to form a seed stock which segregates approx. 3:1 normal: "slender" seedlings. Seed stocks were stored at - 2 0 ~ C. Identification of slender seedlings. Grain was surface-sterilized in 1% sodium hypochlorite for 30 min, washed in sterile water, soaked in filter-sterilized 10 m M HC1 for 10 min, and washed several times in sterile water. It was placed on moist sterile filter paper in plastic trays (approx. 400 mm x 250 mm), covered with plastic film, left at 4 ~ for 4-5 d, then moved to a laboratory bench at approx. 23 ~ C and exposed to ambient lighting. After a further 3-4 d there was maximum discrimination between normal and slender seedlings: the shoots of the latter were approx. 2-2.5 times as long as those of the former (80 mm versus 30 ram), were thinner, and were also a paler green. Identification of endosperm half-grains corresponding to slender seedlings. Dry grains were cut in half transversely with a scalpel to generate two half-grains. The one containing the embryo will be called the "embryo half-grain", the other the "endosperm half-grain". Endosperm half-grains were stored in wells of a microtitre tray, and the embryo half-grains were placed on moist filter paper in a corresponding 12 by 8 matrix. After 4-5 d at 4 ~ C and 3-4 d at 20 ~ C the slender phenotype of the seedlings was scored and the corresponding endosperm halfgrains pooled.

Incubation of half-grains and e-amylase assays. Ten to twelve endosperm half-grains (surface-sterilized as described above) were incubated in 2 ml of 10 mM CaClz with or without hormone for varying times. Half-grains plus medium were transferred to a cold mortar and ground with a pestle in the presence of a small amount of acid-washed sand. The slurry was transferred to a microfuge tube, centrifuged at 12 000. g for 5 min, the supernatant recovered and made 0.05% (w/v) in sodium azide. e-Amylase was assayed using Phadebas tablets (Pharmacia Diagnostics AB, Uppsala, Sweden) as described by Armstrong et al. (1982). In each assay, 15 mg washed freeze-dried Phadebas powder, 100 i11 of 100 m M Na-acetate, pH 5.2, 20 mM CaC12 and 100 ~tl of suitably diluted crude e-amylase preparation were used. After 15 min at 37 ~ C the reaction was stopped by addition of 2.8 ml 0.1 M NaOH, the suspension centrifuged at 1500.g for 3 min and the A62 o of the supernatant determined. Each assay contained approximately 2.5 A62o units of bound chromogene, but dilutions were made so that < 0.25 A62 o units of chromogene were released by the enzyme. Levels of e-amylase are expressed as A62 o units per 15-rain assay per half-grain. Treatment of seedlings with ABA or dehydration. Grains were surface-sterilized and cold-treated as described above and fol-

lowing 3 d at 23 ~ C seedlings were transferred to a desiccator containing 25% (v/v) glycerol (1.5 1 in a 25-cm-diameter desiccator, providing an atmosphere of relative humidity 85-90%), or transferred to filter paper moistened with ABA (10-4 M) in plastic trays, or maintained on water-soaked filter paper in their original trays. After a further 48 h, roots were cut off the seedlings and frozen in liquid nitrogen until use.

Extraction and hybridization of RNA. R N A was extracted from endosperm half-grains as described previously for aleurone layers (Higgins et al. 1976), and from roots of seedlings as described previously for pea cotyledons (Chandler et al. 1983) except for the omission of the high-salt precipitation. For hybridizations, equal amounts (10 gg) of total R N A were subjected to electrophoresis in formaldehyde-agarose gels and blotted to nitrocellulose membrane in 20 • SSC (SSC= 0.15 M NaC1, 0.015 M Na3-citrate). The filters were baked at 80~ for 2 h in a vacuum oven, boiled for 5 min in 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris) pH 8.0, and prehybridized, hybridized, and washed as described in Chandler et al. (1983). The plasmid used as probe (pHVA39) is derived from an ABA-inducible m R N A in aleurone layers of Himalaya barley (Ariffin 1987); the same or closely-related mRNAs are also found in aleurone following dehydration of seedlings (Chandler et al. 1987).

Primer extension analysis of e-amylase transcripts. Synthetic 50-mer oligonucleotides were prepared which are complementary to the clone E sequence (Rogers and Milliman 1983) and to pHV19 (Chandler et al. 1984) from eleven nucleotides prior to the A T G initiation codon and extending 39 nucleotides into the region corresponding to the signal peptide. These primers are specific for low-pI and high-pI e-amylase transcripts, respectively. They were 5'-end-labelled with adenosine 5'-[73ZP]triphosphate and polynucleotide kinase, purified by electrophoresis in denaturing (42%, w/v, urea) 12% polyacrylamide gels and eluted from the gel by the methods described in Maxam and Gilbert (1980). The radioactive primers (approx. 100 fmol) were hybridized to 10 Ixg total R N A at 57~ in 10 gl of 120 mM KC1, 75 mM Tris, pH 8.3. After 60 min, hybridized primers were extended by the addition to each tube of 5 ~tl of reverse transcription components, which, per 50 ~tl, contained 20 gl 0.1 M MgC12,2 gl 1 M dithiothreitol, 10 gl of a mixture of deoxyribonucleoside 5'-triphosphates (10 mM in each of dATP, dCTP, dGTP and dTTP), 16 gl HzO and 2 gl avian myeloblastosis virus (AMV) reverse transcriptase (10 U/gl, Life Science, St Petersburg, Fla., USA). These reactions were carried out at 41 ~ C for 60 min, then to each tube was added 10 gl dye-formamide, the samples were heated at 100 ~ C for 3 rain, then subjected to electrophoresis on denaturing 8% polyacrylamide gels (Maxam and Gilbert 1980) and extended primers visualized by autoradiography using an intensifying screen. The amount of primer used in each hybridization reaction represents at least a 10-fold molar excess over the complementary sequence in the RNA.

Results a-Amylase production

by endosperm half-grains.

Isolated aleurone layers or endosperm half-grains of barley generally produce very little a-amylase in the absence of added GA; however, exceptions have been described where environmental conditions during the grain-filling period may result in

P.M. Chandler: Hormones and gene expression in slender barley

Table 1. Production of a-amylase by WT and "slender" barley endosperm half-grains a

6 0 .o ~

50-

ca -~ E

'~i O

. r-

23

117

~

~

~

~

~.o

,,

Genotype

z 40-

Treatment

0~-Amylase production

A3

z/

0h

24 h

48 h

0.13 -

0.16 32 0.47 0.12 24.5 33.8 2.2 1.9

zz 30-

/

2010

/ -

/

0

o"

9

WT

(Sin~Sin/Sin)

/

/

G A 3 + A .BA 4 k l t "

§

/

~O

0 a,- . . . . .

z' /

z

~-

----II -~"

~..o" un,

f*

-"

Slender

(sin~sin/sin)

j Control 9

I

I

t

I

1

2

3

4

Days of incubation Fig. 1, c~-Amylase activity produced by endosperm half-grains of WT Herta barley grains. Half-grains (10-12 in 2 ml 10 m M CaC12) were incubated at approx. 24 ~ C for the indicated time in control medium (no added hormone), or in medium containing GA3 (1 I~M) or GA3 and ABA (1 gM and 25 gM, respectively). Total e-amylase activity (medium plus tissue) was determined as described in Materials and methods

endosperm c~-amylase production in the absence of added GA3 (Nicholls 1982). Production of c~amylase by endosperm halves of WT Herta grains was found to be highly dependent on added GA3, and this increase was largely prevented by the simultaneous presence of ABA (Fig. 1). The small amount of c~-amylase produced at later stages in the presence of ABA may relate to instability or metabolism (Dashek et al. 1979) of the hormone. Production of 0~-amylase by "slender" endosperm half-grains was assessed after incubation under the same conditions as in the experiment of Fig. 1. "Slender" endosperm half-grains, unlike the WT, produced high levels of c~-amylase in the absence of added GA3 (Table 1). Presence of GA3 resulted in slightly enhanced 0~-amylase production by "slender" half grains and greatly enhanced production by WT half-grains. In both genotypes, ABA was effective in severely reducing c~-amylase production, although "slender" half-grains produced more 0c-amylase than did WT half-grains. The recessive nature of the slender mutation in terms of s-amylase production was investigated by comparing the amounts of enzyme produced by WT endosperm half-grains (genotype of the triploid endosperm Sin~Sin/Sin), and by the "slender" (genotype sin/sin/sin) and " n o r m a l " endosperm half-grains from seeds of heterozygous plants. In the pool of " n o r m a l " endosperm halfgrains there will be equal proportions of three genotypes, Sin/Sin~Sin, Sin~Sin~sin and Sln/sln/sln. If either of the two heterozygote classes produces ~-

Control GA3 GA3 + A B A ABA

-

0.16 2.12 0.16 0.11

Control GA3 GA3 + ABA ABA

0.09 -

2.5 4.7 0.30 0.24

a c~-Amylase activity (A6z o units. (15 min)- 1. (half-grain)- 1) produced by endosperm half-grains of Herta barley incubated with GA3 (1 gM), ABA (25 gM) or without added hormone Table 2. Production of a-amylase by barley endosperm halfgrains enriched for heterozygotes" Genotype

Treatment

c~-Amylase production

Wild type (Sin~Sin~Sin)

Control GA3

0.23 37.5

" N o r m a l " (Sin/Sin~Sin, Sin~Sin~sin, Sin/sin~sin) "Slender" (sin~sin/sin)

Control GA3

0.17 47.5

Control GAa

51 50.3

" e-Amylase activity (A62 o units. (15 9 a. (half-grain)- a) produced by endosperm half-grains of Herta barley incubated for 3 d without added hormone or with GA3 (1 #M)

amylase in the absence of added GA3 the "normal" endosperm half-grains will have significantly higher levels of enzyme production than the WT despite being a genetically heterogeneous population. The results (Table 2) confirm that the recessive phenotype of the "slender" mutation seen in terms of plant growth is also seen at the level of 0~-amylase production in the absence of GA3 (0.17 units for the " n o r m a l " class versus 0.23 units for the WT).

Response of "slender" seedlings to ABA and dehydration. The results in Fig. I and Table 1 indicate that the aleurone of "slender" grains is still responsive to ABA despite its production of s-amylase in the absence of added GA3. This finding is of interest in view of the positive and negative effects of GA and ABA, respectively, on transcription of the 0~-amylase gene (Jacobsen and Beach 1985; Zwar and Hooley 1986). To investigate the response of embryo-derived organs of "slender" plants to ABA, "slender" seedlings were placed

118

P.M. Chandler: Hormones and gene expression in slender barley

Fig. 2. Hybridization of a cDNA clone (pHVA39) for an ABAinducible m R N A to size-fractionated RNA from roots of wild type and "slender" barley seedlings. Lanes a-c contain RNA from roots of WT seedlings (a: 3 d water+2 d water, b: 3 d w a t e r + 2 d 1 0 - ~ M ABA, c: 3 d w a t e r + 2 d dehydration). Lanes d-f are the same treatments as a-c, in the same order, but contain RNA from roots of slender seedlings. Lanes g, h represent a longer exposure of lanes d, e. In each lane there was 10 gg total R N A initially loaded on the gel. The arrow denotes the position of the m R N A (approx. 1200 bases) hybridizing to pHVA39 in RNA from ABA-treated barley aleurone

on an ABA solution for 48 h, or dehydrated for 48 h in an atmosphere of 85-90% relative humidity. The level of m R N A detected by pHVA39 in total RNA from roots of normal seedlings, seedlings transferred to ABA for 48 h, and seedlings exposed to dehydration was examined by hybridization (Fig. 2). In both "slender" and WT seedlings the level of mRNAs hybridizing with pHVA39 increased substantially above controls following treatment with exogenous ABA or following dehydration, indicating that gene expression is being regulated by ABA in the "slender" seedlings as it is in WT seedlings. Both of these treatments would be expected to result in elevated levels of endogenous ABA (Wright and Hiron 1969). o~-Amylase m R N A s in W T and "slender" caryopses. The expression of c~-amylase production by "slender" endosperm half-grains (Tables 1, 2) indicates that there is an increase in the levels of the e-amylase mRNAs occuring in the aleurone of such grains. In order to determine whether the types of c~-amylase mRNAs expressed by the "slender" grains were the same as expressed by WT grains in the presence of GA3, primer extension experiments were carried out. Using this technique it has been possible to define at least two different mRNAs in each of the low-pI and high-pI families of c~-amylase isoenzymes of Himalaya barley aleurone (Chandler et al. 1987). Extended primers from the cultivar Herta are very similar to those from the cultivar Himalaya except for the absence of the longest extended

Fig. 3. Primer extensions on RNAs from barley endosperm. Aliquots of RNA (10 gg) were hybridized with 32p-labelled oligonucleotide primers specific for either the low-pI (upper panel) or high-pI (lower panel) a-amylase families. Primers were extended using AMV reverse transcriptase, fractionated on denaturing 12% acrylamide gels and visualized by autoradiography. The numbers denote the principal m R N A members of each family (defined in Himalaya barley), and correspond to the length in bases of the 5' untranslated region of the mRNA. The RNA was derived from endosperm of: Himalaya seedlings (3 d post-imbibition, lane a), WT Herta seedlings (2 d post-imbibition, lane b), WT Herta half-grains incubated 2 d - G A (lane c) or 2 d + GA (lane d), and "slender" Herta half-grains incubated 1, 2, 3 or 4 d - G A (lanes e-h). The relative levels of extended primers reflect the level of corresponding m R N A

primer in the high-pI family (Fig. 3); although not clearly visible in lane b of Fig. 3, both of the low-pI extended primer bands are present. Within each family (low-pI or high-pI) no qualitative differences were seen between R N A prepared from the endosperm of seedlings, WT endosperm halfgrains in the presence of GA3, or "slender" halfgrains in the absence of GAa. However, there were large differences in the total amount of extended primers seen in RNA from WT half-grains in the presence or absence of GA3, and in R N A from "slender" half-grains 1, 2, 3 or 4 d after imbibition. These results indicate that regulation of expression of c~-amylase is occurring at the m R N A level, as hybridization is carried out under conditions of primer excess and therefore the amount of each extended primer depends upon the amount of corresponding mRNA. Furthermore, the profile of mRNAs expressed by "slender" half-grains in the absence of GAa is identical to that expressed by WT half-grains in the presence of GA3. These

P.M. Chandler: Hormones and gene expression in slender barley

patterns in turn are the same as seen in grain following germination. Discussion

While ~ slender" plants do not set seed, it was possible to study the aleurone response of the slender genotype by germinating embryo half-grains from heterozygous plants and, based on the appearance of the slender phenotype in the seedling, pooling endosperm half-grains which corresponded to the slender seedlings. "Slender" endosperm halfgrains produced almost as much 0~-amylase in the absence of GA3 as in its presence. This behaviour contrasts that of the WT which showed an almost absolute requirement for added GAa before 0~amylase was produced. Identical sets of low-pI and high-pI 0~-amylase mRNAs were found by primer extension assays in the endosperm RNA of seedlings, WT half-grains treated with GA3 or slender half-grains in the absence of GA3. The slender mutation therefore mimics the GA response in terms of production of both c~-amylase mRNA and the enzyme. In both genotypes (mutant and WT) 0~-amylase production was prevented by ABA. Furthermore, the roots of both slender and WT seedlings responded to applied ABA and to dehydration by accumulation of an ABA-inducible mRNA. These results indicate that the homozygous slender genotype retains sensitivity to ABA. Following germination of normal barley grains a signal from the embryo is responsible for aleurone production of c~-amylase. There is circumstantial evidence for GA being the signal. For instance, bioassay data indicate that the scutellum is a major source of GA (possibly GA1) in the first days of seedling growth (Radley 1967). A study using combined gas chromatography-mass spectrometry has shown GA1 to be the major GA species present in barley grains following germination (Yamada /982). Finally there is the well-known production of 0~-amylase by de-embryonated grains or isolated aleurone layers in response to applied GA (Paleg 1960; Yomo 1960). The results presented in this paper are consistent with the view that GA is responsible for aleurone c~-amylase production since the slender mutant, which in its vegetative parts exhibits a near-maximal GA response, also shows such a response in endosperm tissue. However, the evidence favouring GA as the signal is still indirect. Dwarf mutants of corn and pea have been described which lack GA~ (Spray et al. 1984; Ingram et al. 1984). If similar mutants were available in barley it might be possible to test in a more direct

119

manner the proposal that scutellar production of GA1 is responsible for production of c~-amylase in the aleurone. The biochemical defect in slender plants responsible for the mutant phenotype has not been identified. At least two simple models to explain the phenotype can be postulated: firstly, slender plants may, as a consequence of altered GA biosynthesis or metabolism, contain high endogenous levels of active GAs. Secondly, they may contain normal levels of GA but be altered in their sensitivity such that a near-maximal GA response is seen in the presence of normal GA levels (and perhaps even in the absence of GA). A variant of these models involves normal GA levels but altered cellular or intracellular distribution, so that targets for GA action effectively are exposed to higher levels of GA. In pea (Pisum sativurn L.) a similar "slender" phenotype has been described, and in this case introduction of mutations which block GA biosynthesis (le and na) has no effect on expression of the "slender" phenotype despite their effect on GA metabolism (Potts et al. 1985; Ingram and Reid J987). A further interesting aspect of the "slender" mutation is that it behaves as a recessive character both in terms of whole-plant phenotype (Foster 1977) and in c~-amylase production (Table 2). A mutation conferring expression of a character (0~amylase production) in the apparent absence of the normal inducer (GA) might be expected to show partial expression in the heterozygote if it involved over-production of a positive effector. The fact that no expression was found in the heterozygote indicates that the slender mutation may interfere with the activity of a negative effector of a-amylase gene expression. As long as heterozygotes contained sufficient activity of the WT allele, negative regulation would still occur. It is of interest to note that steroid hormones have recently been shown to relieve negative control of the chicken ovalbumin promoter (Gaub et al. 1987). The most advanced understanding of molecular aspects of GA action comes from studies in barley aleurone. It is apparent from the results described here that the aleurone of the slender mutant resembles WT aleurone treated with GA. This indicates that the slender mutant will provide a good model for analysis of the action of GA in vegetative parts of barley plants. I am very grateful to Chris Foster for providing seed of the slender m u t a n t and much useful information on its characteristics. I would like to t h a n k T.J. Close and R. Hooley for their comments on the manuscript.

120

References Ariffin, Z. (1987) Regulation of protein synthesis by ABA and PA in barley aleurone layers. M.Sc. thesis, Australian National University, Canberra, A.C.T., Australia Armstrong, C., Black, M., Chapman, J.M., Norman, H.A., Angold, R. (1982) The induction of sensitivity to gibberellin in aleurone tissue of developing wheat grains. I. The effect of dehydration. Planta 154, 573-577 Chandler, P.M., Higgins, T.J.V., Randall, P.J., Spencer, D. (1983) Regulation of legumin levels in developing pea seeds under conditions of sulfur deficiency. Plant Physiol. 71, 47-54 Chandler, P.M., Jacobsen, J.V., Zwar, J.A., Ariffin, Z., Huiet, L. (1987) Control of a-amylase gene expression by gibberellin and abscisic acid in barley aleurone. In: Molecular Biology of Plant Growth Control (UCLA Syrup. on Molecular and Cellular Biology) pp. 22-33 Fox, J.E., Jacobs, M., eds. Alan R. Liss, New York, N.Y., USA Chandler, P.M., Zwar, J.A., Jacobsen, J.V., Higgins, T.J.V., Inglis, A.S. (1984) The effects of gibberellic acid and abscisic acid on a-amylase mRNA levels in barley aleurone layers: studies using an a-amylase cDNA clone. Plant Mol. Biol. 3, 407-418 Dashek, W.V., Singh, B.N., Walton, D.C. (1979) Abscisic acid localization and metabolism in barley aleurone layers. Plant Physiol. 64, 43-48 Foster, C.A. (1977) Slender: an accelerated extension growth mutant of barley. Barley Genet. Newslett. 7, 24-27 Gaub, M., Dierich, A., Astinotti, D., Touitou, I., Chambon, P. (1987) The chicken ovalbumin promoter is under negative control which is relieved by steroid hormones. EMBO J. 6, 2313-2320 Higgins, T.J.V., Zwar, J.A., Jacobsen, J.V. (1976) Gibberellic acid enhances the level of translatable mRNA for a-amylase in barley aleurone layers. Nature 260, 166-169 Ingram, T.J., Reid, J.B. (1987) Internode length in Pisum: Biochemical expression of the le and na mutations in the slender phenotype. J. Plant Growth Regul. S, 235-243 Ingrain, T.J., Reid, J.B., Muffet, I.C., Gaskin, P., Willis, C.L., MacMillan, J. (1984) Internode length in Pisum. The Le gene controls the 3fl-hydroxylation of gibberellin Azo to gibberellin A~. Planta 160, 455-463 Jacobsen, J.V., Beach, L. (1985) Control of transcription of a-amylase and rRNA genes in barley aleurone protoplasts by gibberellin and abscisic acid. Nature 316, 275-277 Maxam, A.W., Gilbert, W. (1980) Sequencing and-labelled

P.M. Chandler: Hormones and gene expression in slender barley DNA with base-specific chemical cleavages. Meth. Enzymol. 65, 488-560 Moore, R., Smith, J.D. (1985) Graviresponsiveness and abscisic acid content of roots of carotenoid-deficient mutants of Zea mays L. Planta 164, 126--128 Neill, S.J., Horgan, R., Parry, A.D. (1986) The carotenoid and abscisic acid content of viviparous kernels and seedlings of Zea rnays L. Planta 169, 87-96 Nicholls, P.B. (1982) Influence of temperature during grain growth and ripening of barley on the subsequent response to exogenous gibberellic acid. Aust. J. Plant Physiol. 9, 373-383 Paleg, L.G. (1960) Physiological effects of gibberellic acid. II. On starch hydrolyzing enzymes of barley endosperm. Plant Physiol. 35, 902-906 Potts, W.C., Reid, J.B., Muffet, I.C. (1985) Internode length in Pisum. Gibberellins and the slender phenotype. Physiol. Plant. 63, 357-364 Radley, M. (1967) Site of production of gibberellin-like substances in germinating barley embryos. Planta 75, 164-171 Rogers, J.C., Milliman, C. (1983) Isolation and sequence analysis of a barley e-amylase cDNA clone. J. Biol. Chem. 258, 8169-8174 Spray, C., Phinney, B.O., Gaskin, P., Gilmour, S.J., MacMillan, J. (1984) Internode length in Zea mays L. The dwarf-1 mutation controls the 3fl-hydroxylation of gibberellin Azo to gibberellin Aa. Planta 160, 464-468 Tal, M. (1966) Abnormal stomatal behaviour in wilty mutants of tomato. Plant Physiol. 41, 1387-1391 Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201 5205 Yamada, K. (1982) Determination of endogenous gibberellins in germinating barley by combined gas chromatographymass spectrometry. J. Am. Soc. Brew. Chem. 40, 18-25 Wright, S.T.C., Hiron, R.W.P. (1969) (+)-Abscisic acid, the growth inhibitor induced in detached wheat leaves by a period of wilting. Nature 224, 719-720 Yomo, H. (1960) Studies on the a-amylase activating substances. IV. On the amylase activating action of gibberellin. Hakko Kyokaichi 18, 600-602 Zwar, J.A., Hooley, R. (1986) Hormonal regulation of a-amylase gene transcription in wild oat (Avenafatua L.) aleurone protoplasts. Plant Physiol. 80, 459-463 Received 14 October 1987; accepted 8 February 1988

Hormonal regulation of gene expression in the "slender" mutant of barley (Hordeum vulgare L.).

The "slender" mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA3). Expression of GA3-regulated and absc...
850KB Sizes 0 Downloads 0 Views