| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiol. (1998) 118: 1495-1506
Expression of
|
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Alfalfa (Medicago
sativa L.) roots contain large quantities of 
-amylase, but
little is known about its role in vivo. We studied this by isolating a
-amylase cDNA and by examining signals that affect its expression.
The -amylase cDNA encoded a 55.95-kD polypeptide with a deduced
amino acid sequence showing high similarity to other plant
-amylases. Starch concentrations, -amylase activities, and
-amylase mRNA levels were measured in roots of alfalfa after defoliation, in suspension-cultured cells incubated in sucrose-rich or
-deprived media, and in roots of cold-acclimated germ plasms. Starch
levels, -amylase activities, and -amylase transcripts were
reduced significantly in roots of defoliated plants and in sucrose-deprived cell cultures. -Amylase transcript was high in
roots of intact plants but could not be detected 2 to 8 d after defoliation. -Amylase transcript levels increased in roots between September and October and then declined 10-fold in November and December after shoots were killed by frost. Alfalfa roots contain greater -amylase transcript levels compared with roots of
sweetclover (Melilotus officinalis L.), red clover
(Trifolium pratense L.), and birdsfoot trefoil
(Lotus corniculatus L.). Southern analysis indicated
that -amylase is present as a multigene family in alfalfa. Our
results show no clear association between -amylase activity or
transcript abundance and starch hydrolysis in alfalfa roots. The great
abundance of -amylase and its unexpected patterns of gene expression
and protein accumulation support our current belief that this protein
serves a storage function in roots of this perennial species.
The mode of regulation of plant Alfalfa (Medicago sativa L.) is an excellent system in which
to study mechanisms of starch utilization and accumulation. In agricultural ecosystems alfalfa is completely defoliated at
approximately 30-d intervals. Rapid herbage regrowth after defoliation
has been positively associated with quantities of carbon and nitrogen
reserves in taproots, including starch (Graber et al., 1927 Nitrogen-containing compounds in alfalfa roots (such as amino acids and
proteins) have been shown to be positively associated with the rate of
herbage regrowth after defoliation (Kim et al., 1991 In view of the great abundance of Plant Material Used for Isolation of
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-Amylase catalyzes the hydrolysis of 
-1,4-glucosidic
linkages from the nonreducing ends of starch molecules releasing
maltose and producing -limit dextrin (Thomas et al., 1971
). It is
abundant in seeds and roots of certain species (Doehlert et al., 1982; Yoshida and Nakamura, 1991; Boyce and Volenec, 1992a) and is also present in other vegetative tissues (Beck and Ziegler, 1989). Because of its high abundance and its perceived role in starch metabolism, -amylase has been the focus of several physiological and
molecular studies. Molecular analyses of plant -amylase have been
conducted using cDNAs or genomic sequences from both dicots (Monroe et
al., 1991; Yoshida and Nakamura, 1991; Totsuka and Fukazawa, 1993) and
monocots (Sadowski et al., 1993; Yoshigi et al., 1994; Wagner et
al., 1996; Wang et al., 1997). Encoded amino acid sequences for these
plant -amylases are highly conserved, with amino acid similarity
ranging from 60% to 96%. An endosperm-specific -amylase has been
described for rye (Rorat et al., 1991) and barley (Yoshigi et al.,
1994) that contains Gly-rich repetitive sequences in the carboxyl
terminus of the protein.
-amylase genes appears complex and
at times contradictory. In many plant systems -amylase transcript
accumulation is regulated by sugars. Arabidopsis -amylase mRNA
levels increased in rosette leaves when plants or excised, fully
expanded leaves were supplied with Suc, Glc, and Fru but were not
affected by mannitol or sorbitol (Mita et al., 1995). Exposure of
Arabidopsis plants to light was essential for the accumulation of the
-amylase transcript. Light also induced accumulation of the
-amylase transcript in mustard cotyledons (Sharma and Shopfer,
1987). Sweet potato -amylase gene expression occurs in darkness if
leaf-petiole cuttings are supplied with Suc (Nakamura et al., 1991).
Dipping sweet potato leaf-petiole cuttings in polygalacturonic acid or
chitosan also induced -amylase mRNA accumulation, whereas mechanical
wounding of leaves only occasionally induced -amylase gene
expression (Ohto et al., 1992). ABA induced the expression of sweet
potato -amylase in leaf-petiole cuttings within 12 h of
treatment (Ohto et al., 1992). However, in rice aleurone cells, ABA
inhibited de novo synthesis of -amylase and reduced -amylase transcript levels (Wang et al., 1996). In most systems studied to date
increases in -amylase activity and accumulation of -amylase transcripts were associated with starch deposition in tissue. This
raises questions regarding the in vivo role of plant -amylase as a
starch hydrolase.
; Smith,
1962; Hendershot and Volenec, 1993b). Previous reports showed that
defoliation results in a decline in both root amylase activity (>99%
-amylase) and starch concentration (Volenec and Brown, 1988; Volenec
et al., 1991). Roots of other forage legumes such as sweetclover (Melilotus officinalis L.), red clover (Trifolium
pratense L.), and birdsfoot trefoil (Lotus corniculatus
L.) contain about one-half (red clover) or less of the -amylase
activity levels found in alfalfa roots (Li et al., 1996). Like alfalfa,
roots of sweetclover and red clover exhibit a decline in -amylase
activity that parallels a decline in root starch concentration after
defoliation (Li et al., 1996). Very low total amylase specific activity
was observed in roots of birdsfoot trefoil, even though root starch was
depleted in a manner similar to that of alfalfa after defoliation
(Boyce et al., 1992). Clearly, the pattern of decreased -amylase
activity coincident with large declines in root starch concentration
are inconsistent with the perceived role of -amylase as a starch hydrolase in roots of alfalfa and the other forage legumes.
; Hendershot and
Volenec, 1993b; Ourry et al., 1994; Barber et al., 1996; Volenec et
al., 1996) and in the spring when shoot growth resumes (Volenec
et al., 1991; Hendershot and Volenec, 1993a; Li et al., 1996). Three
polypeptides constituting approximately 40% of the root's soluble
protein pool have been isolated and characterized (Cunningham and
Volenec, 1996). We believe that these polypeptides are VSPs because
they accumulate in alfalfa taproots in early autumn and disappear in
the spring and after plants are defoliated, in a manner that is
consistent with functions assigned to VSPs (Cyr and Bewley, 1990).
Boyce and Volenec (1992b) purified a 57.5-kD -amylase protein from
alfalfa taproots and found that it constituted 8% of root-soluble
protein. The seasonal pattern of -amylase activity followed the
trends in concentration of root VSPs, increasing in autumn and
declining markedly in spring (Volenec et al., 1991; Hendershot and
Volenec, 1993a; Li et al., 1996). Because defoliation reduces
nitrogenase activity (Vance et al., 1979) and uptake of nitrogen from
the soil (Kim et al., 1993), we speculate that -amylase, like VSPs,
may be hydrolyzed to its constituent amino acids, which are then
transported to regrowing shoots to provide some of the nitrogen needed
for herbage growth in spring and regrowth after defoliation in the
summer (Boyce and Volenec, 1992b).
-amylase in alfalfa roots and its
unexpected pattern of activity during root starch loss, it is important
to understand how -amylase gene expression is regulated by
environmental cues such as defoliation and cold temperatures. Our
objectives were: (a) to isolate a cDNA for alfalfa root -amylase; (b) to determine tissue-specific expression of the -amylase gene; (c) to examine the extent to which roots of other perennial forage legumes accumulate -amylase mRNA; (d) to determine genomic
organization of the -amylase gene; and (e) to determine how
defoliation, Suc deprivation in cell-suspension cultures, and winter
hardening influence -amylase transcript levels, -amylase
activity, and root starch concentrations.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-Amylase
cDNA
-amylase cDNA isolation. Seedlings were established at the Agronomy Research Center of Purdue
University in April. Randomization of field plots and management practices were as described previously (Cunningham et al., 1998). Plants were defoliated in mid-August, and roots were sampled on October
15. Roots were washed free of soil under a stream of cold water.
Nodules were removed and discarded to ensure root tissue specificity.
The top 5 cm of the roots were immersed in liquid nitrogen, packed in
solid CO2, and transported to the laboratory, where tissues were stored at 
80°C.
Total RNA Extraction
Total RNA was isolated using hot phenol and the procedure of Ougham and Davis (1990) with minor modifications.
Polyvinylpolypyrrolidone (0.4 g) was added to tubes (Nalgene)
containing 2 g of frozen root tissue that had been ground in
liquid nitrogen. Water-saturated phenol (8 mL, 65°C) was added to
each tube and the tubes were placed in a 65°C water bath. When
samples thawed, 8 mL of RNA-extraction buffer (0.2 M sodium
acetate, pH 5.2, 0.01 M EDTA, 1% SDS) was added to each
tube, and the samples were incubated at 65°C for 15 min. The tubes
were removed from the 65°C water bath and cooled to 25°C, and 8 mL
of chloroform was added. Agitation of tubes, centrifugation, and
recovery of the RNA pellet was as described by Ougham and Davis (1990).
Isolation and Identification of Alfalfa -Amylase cDNA
-amylase cDNA, the first-strand cDNA
was synthesized using 0.2 µM of a modified
oligo(dT)-primer, 5
-GAGAAGCT12GC-3, and 200 units of reverse transcriptase (Superscript II, BRL/Life Technologies)
according to the manufacturer's protocol. The total volume for the
reaction was 20 µL. White clover (Trifolium repens L.) RNA
was used as an internal control because the upstream primer used for
reverse transcriptase-PCR amplification of putative alfalfa -amylase
cDNA clones had been made to sequences of the 5 region of white clover
-amylase cDNAs (accession no. AF049098). PCR amplification was in a
50-µL reaction volume containing 2 µL of the first-strand cDNA
mixture, 1× PCR buffer, 0.2 mM final concentration of the
deoxyribonucleotide triphosphates, 0.2 µM final
concentration of the upstream primer (5-CAA, GGC, CAC, TTC, TAA, CAA,
CAT, G-3), 0.2 µM final concentration of the modified oligo(dT)-primer (5-GAGAAGT12GC-3), and 5 units
of Taq DNA polymerase. The temperature profile for the
thermocycler was 94°C for 5 min, 40 cycles at 94°C for 30 s,
50°C for 30 s, 72°C for 2 min, and then 75°C for 15 min
after PCR. The PCR products were analyzed on a 1.5% agarose gel. Four
bands with approximate sizes of 635, 969, 1345, and 1701 bp were eluted
from the gel and subcloned into the pGEM-T vector (Promega). The 1.7-kb
band from alfalfa roots also corresponded to a band of similar size
from white clover and was further analyzed.
DNA Sequencing and Analysis
A plasmid containing the 1.7-kb putative-amylase cDNA
designated pMSBA1 (M.
sativa
-amylase 1) was digested with
HindIII and liberated an approximately 800-bp 3 fragment.
This fragment was subcloned into pBluescript SK() plasmid (Stratagene). The two subclones (pMSBA1 minus the HindIII
fragment and pBluescript plus the HindIII fragment) were
sequenced on both strands using an automated DNA sequencer (Pharmacia)
with both universal and specific primers. Homology searches were
obtained with the Basic Local Alignment Search Tool of the National
Center for Biotechnology Information (Altschul et al., 1990). Predicted amino acid sequences were analyzed using software from the Genetics Computer Group (Madison, WI).
Tissue- and Species-Specific Expression of -Amylase
) and planted in 1-L plastic pots containing a 1:1 mixture of
silt loam soil:sand. Pots were placed in a greenhouse in which air
temperature was 25°C ± 5°C and the photoperiod was extended
to 15 h with fluorescent and incandescent lights (160 µmol
m
2 s1). Plants were
watered as needed with water purified by reverse osmosis and were also
provided with 50 mL of nitrogen-free Hoagland solution twice
weekly. Plants were grown to the flowering stage. To determine the
tissue-specific expression of -amylase in alfalfa, the leaves,
stems, and roots (without nodules) were sampled in three replicates. To
determine species-specific expression of -amylase, three replicates
of root tissues of each of the five forage legume species were sampled
at the time of flowering. Tissues were processed and stored for RNA
analysis as described above.
Effects of Defoliation on Starch Accumulation and -Amylase
Expression
-amylase expression, one-half of the alfalfa plants were defoliated,
leaving a 5-cm stubble. Roots were sampled immediately (0 h), at 3, 6, and 12 h, and at 1, 2, 4, 8, 12, 16, 20, 24, and 28 d after
defoliation. Herbage of the remaining alfalfa plants was left intact to
serve as undefoliated controls, and roots of these plants were sampled
at the same time as the defoliated plants. Roots were washed free of
soil under a stream of cold water. Nodules were removed and discarded.
The top 5 cm of taproots was selected for analysis to avoid errors due
to plant-to-plant variations in root length. Previous studies have
shown that results obtained using the uppermost 5 cm of taproot were
closely associated with results obtained using the reminder of the root
system (J.J. Volenec, unpublished data). Root tissues were immersed in
liquid nitrogen, packed in solid CO2, and taken
to the laboratory, where tissues were stored at 80°C. The remaining
root tissue was lyophilized, ground to pass a 1-mm screen, and
stored at 20°C until analyzed for starch concentration and
-amylase activity. In this experiment pots were arranged in a
randomized, complete block design with three replications. For
statistical analysis, variation was partitioned into replicate,
defoliation treatment, sampling time after defoliation, and
corresponding interactions.
Influence of Suc Deprivation on Starch Accumulation and -Amylase
Expression in Cell Suspensions
-amylase transcript abundance in other
plant systems, we were interested in using cell-suspension cultures to
alter the sugar availability to alfalfa cells and study its impact on
-amylase gene expression. Alfalfa cv Pioneer Brand 5929 seeds were
surface-sterilized in 20% (v/v) commercial bleach for 10 min and
washed three washes with sterile water. Seeds were germinated on
filter-paper bridges at 25°C under a 16-h photoperiod of fluorescent
lights (25 µmol m2
s1) for 7 d. Callus was initiated from
5-cm-long root sections on Murashige-Skoog medium (Murashige and Skoog,
1962) supplemented with 1 mg L1 2,4-D. Gamborg
et al. (1968) B5g liquid medium supplemented with 1 mg
L1 2,4-D was used for induction of suspension
cultures. Control cells were grown in B5g medium containing 20 g
L1 Suc. For the Suc-depleted B5g media, Suc was
replaced with an equimolar concentration of mannitol. Cells were
sampled 24, 48, and 72 h after transfer to Suc-deprived or normal
B5g media. Cells were recovered from suspensions and immediately frozen
in liquid nitrogen and stored at 80°C for analysis. The
cell-culture experiment was replicated three times, and flasks were
arranged in a randomized, completed block design. For statistical
analysis, experimental variation was partitioned into replicate
effects, Suc treatment effects, sampling time effects, and
corresponding interactions using analysis of variance.
Effects of Fall Hardening on Starch Utilization and -Amylase
Expression in Contrasting Alfalfa Germ Plasms
-amylase expression in closely related alfalfa germ plasms that
possess contrasting fall dormancy and winter hardiness. The six diverse
alfalfa cultivars used for this study included fall nondormant,
nonhardy cv CUF 101-O, fall dormant, winter hardy cv Norseman-O (O = original cultivar), and populations from the third cycle of selection
for contrasting fall dormancy from within these two cultivars
(Cunningham et al., 1998). The contrasting populations of cv Norseman-O
and cv CUF-101-O, cv Norseman-L and cv CUF 101-L for germ plasms
selected for increasing fall dormancy, and cv Norseman-H and cv CUF
101-H were designated for the germ plasms selected for decreasing fall
dormancy. Seedlings were established in April at the Agronomy Research
Center. The top 5-cm root sections were sampled on September 10, October 15, November 18, and December 17, and tissues were handled as
described above for RNA isolation. In addition, the remaining root
tissue was cut into 5-cm segments, packed in solid
CO2, transported to the laboratory, lyophilized, ground to pass a 1-mm screen, and stored at 20°C until analysis. This study was replicated four times. Data were analyzed as a split-plot design with repeated sampling of plants from within rows
over time. Data from starch concentrations and -amylase activity
were analyzed using statistical analysis software (SAS Institute,
1989).
Northern Hybridization Analysis
Total RNA (20 µg) was separated on 1.5% agarose formaldehyde gels (Lehrach et al., 1977) and transferred to Zeta-probe membranes (Bio-Rad). The membranes were prehybridized for 4 h at 42°C
(Stewart and Walker, 1989) with slow shaking. The insert (-amylase
cDNA) from pMSBA1 was labeled with [32P]dCTP
using random priming (Feinberg and Vogelstein, 1983). Hybridization and
washing of membranes were as described by Gana et al. (1997). To
determine the amount of total RNA loaded in each lane, the P-labeled -amylase probe was stripped from
the membranes using the manufacturer's protocol (Bio-Rad) and
rehybridized with a P-labeled alfalfa 18S
ribosomal probe to correct for RNA loading differences. Membranes were
exposed to radiographic film (Fuji, Tokyo, Japan) at 80°C.
Signal intensities were quantified using an imager (Packard
Instruments, Downers Grove, IL).
Genomic Southern Analysis
Genomic DNA was extracted from roots of field-grown alfalfa using a urea-based DNA miniprep procedure (Shure et al., 1983). The DNA (10 µg) was digested separately with HindIII, NdeI,
EcoRI, EcoRV, and BglII. Fragments
were separated on a 1% agarose gel and transferred to Zeta-probe
membranes (Bio-Rad). Membranes were prehybridized and hybridized as
described by the manufacturer (Bio-Rad) using the
32P-labeled -amylase probe described above.
Starch Analysis
For starch analysis, 30 mg of ground, freeze-dried tissue or 100 mg of fresh root tissue ground in liquid nitrogen was used. Sugars were initially extracted using 80% (v/v) ethanol. Starch in the ethanol-extracted residue was analyzed as previously described (Li et al., 1996), except that the -amylase product A-2643 (Sigma) was
substituted for the -amylase product A-0273 (Sigma).
-Amylase Assay
-amylase assay
procedures were conducted at 4°C or on ice unless otherwise
indicated. Soluble protein was extracted from 30 mg of ground,
freeze-dried tissue or 100 mg of fresh root tissue ground in liquid
nitrogen using 1 mL of 100 mM imidazole buffer, pH 6.5, containing 10 mM 2-mercaptoethanol and 1 mM
PMSF. Samples were vortexed three times and centrifuged at
14,000g, and the soluble protein concentration of the
supernatant was estimated using protein dye-binding (Bradford, 1976).
-Amylase activity was determined by diluting the buffer-soluble proteins 1:10 with 100 mM imidazole buffer, pH 7.5, and
incubating 25 µL of the diluted sample with 50 µL of
p-nitrophenol--D-maltopentaose, a specific
-amylase substrate (Betamyl kit, Megazyme International, Bray,
County Wicklow, Ireland). Samples were incubated for 5 min at 40°C
and the reactions were terminated by the addition of 2 mL of 1.65 M Tris, and the A410 was
determined. One unit of activity was defined as the quantity of enzyme
that released 1 µM of p-nitrophenol min1.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Isolation and Characterization of Alfalfa -Amylase
-amylase sequences. The 1.7-kb band was analyzed further because it was judged
to be close to a full-length cDNA, based on our previous molecular
characterization of the -amylase polypeptide (Boyce and Volenec,
1992b).
-amylases. Three potential
polyadenylation signals (Joshi, 1987) were found in the 141-bp
3-untranslated region.
Genome Analysis of the To determine distribution of the
Tissue- and Species-Specific Expression of
Defoliation-Induced Changes in Starch Concentrations,
Levels of Starch,
Effect of Fall Hardening on Starch Concentrations,
We have isolated and sequenced a
Received July 20, 1998;
accepted September 18, 1998.
Abbreviation:
VSP, vegetative storage protein.
Altschul SF,
Gish W,
Miller W,
Myers EW,
Lipman DJ
(1990)
Basic local alignment search tool.
J Mol Biol
215:
403-410
[CrossRef][ISI][Medline]
Avice J-C,
Ourry A,
Volenec JJ,
Lemaire G,
Boucard J
(1996)
Defoliation-induced changes in abundance and immuno-localization of vegetative storage proteins in taproots of Medicago sativa.
Plant Physiol Biochem
34:
1-11
Bairoch A
(1994)
PROSITE: recent developments.
Nucleic Acids Res
22:
3578-3580
Barber LD,
Joern BC,
Volenec JJ,
Cunningham SM
(1996)
Supplemental nitrogen effects on alfalfa regrowth and taproot nitrogen mobilization.
Crop Sci
36:
1217-1223
Beck E,
Ziegler P
(1989)
Biosynthesis and degradation of starch in higher plants.
Annu Rev Plant Physiol Plant Mol Biol
40:
95-117
[CrossRef][ISI]
Boyce PJ,
Penaloza E,
Volenec JJ
(1992)
Amylase activities in roots of Medicago sativa L. and Lotus corniculatus L. following defoliation.
J Exp Bot
43:
1053-1059
Boyce PJ,
Volenec JJ
(1992a)
Taproot carbohydrate concentrations and stress tolerance of contrasting alfalfa genotypes.
Crop Sci
32:
757-761
Boyce PJ,
Volenec JJ
(1992b)
Bradford MM
(1976)
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Cunningham SM,
Volenec JJ
(1996)
Purification and characterization of vegetative storage proteins from alfalfa (Medicago sativa L.) taproots.
J Plant Physiol
147:
625-632
[ISI]
Cunningham SM,
Volenec JJ,
Teuber LR
(1998)
Plant survival and root and bud composition of alfalfa populations selected for contrasting fall dormancy.
Crop Sci
38:
962-969
Cyr DR,
Bewley JD
(1990)
Proteins in roots of the perennial weeds chicory (Cichorium intylus L.) and dandelion (Taraxacum officinale Weber) are associated with overwintering.
Planta
182:
370-374
[CrossRef][ISI]
Doehlert DC,
Duke SH,
Anderson L
(1982)
Beta-amylases from alfalfa (Medicago sativa L.) roots.
Plant Physiol
69:
1096-1102
Feinberg AP,
Vogelstein B
(1983)
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal Biochem
132:
6-13
[CrossRef][ISI][Medline]
Gamborg OL,
Miller NA,
Ojima K
(1968)
Nutrient requirements of suspension cultures of soybean root cells.
Exp Cell Res
50:
151-158
[CrossRef][ISI][Medline]
Gana JA,
Sutton F,
Kenefick DG
(1997)
cDNA structure and expression patterns of a low-temperature-specific wheat gene tacr7.
Plant Mol Biol
34:
643-650
[CrossRef][ISI][Medline]
Graber LH, Nelson NT, Luekel WA, Albert WB (1927) Organic food
reserves in relation to the growth of alfalfa and other perennial
herbaceous plants. Wis Agric Exp Stn Bull 80
Habben JE,
Volenec JJ
(1991)
Amylolytic activity in taproots of diploid and tetraploid Medicago sativa L.
Ann Bot
68:
393-400
Hendershot KL,
Volenec JJ
(1993a)
Taproot nitrogen accumulation and use in overwinter alfalfa (Medicago sativa L.).
J Plant Physiol
141:
68-74
Hendershot KL,
Volenec JJ
(1993b)
Nitrogen pools in roots of Medicago sativa L. after defoliation.
J Plant Physiol
141:
129-135
Joshi CP
(1987)
Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis.
Nucleic Acids Res
15:
9627-9640
Kim TH,
Ourry A,
Boucaud J,
Lemaire G
(1991)
Changes in source sink relationship for nitrogen during regrowth of lucerne (Medicago sativa L.) following removal of shoots.
Aust J Plant Physiol
18:
593-602
Kim TH,
Ourry A,
Boucard J,
Lemaire G
(1993)
Partitioning of nitrogen derived from N2 fixation and reserves in nodulated Medicago sativa L. during regrowth.
J Exp Bot
44:
555-562
Lehrach H,
Diamond D,
Wozney JM,
Booedtker H
(1977)
RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination.
Biochemistry
16:
4743-4751
[CrossRef][Medline]
Li R,
Volenec JJ,
Joern BC,
Cunningham SM
(1996)
Seasonal changes in nonstructural carbohydrates, proteins, and macronutrients in roots of alfalfa, red clover, sweetclover, and birdsfoot trefoil.
Crop Sci
36:
617-623
Lizotte PA,
Henson CA,
Duke SH
(1990)
Purification and characterization of pea epicotyl
Mita S,
Hirano H,
Nakamura K
(1997)
Negative regulation in the expression of a sugar-inducible gene in Arabidopsis thaliana.
Plant Physiol
114:
575-582
[Abstract]
Mita S,
Suzuki-ujii K,
Nakamura K
(1995)
Sugar-inducible expression of a gene for beta-amylase in Arabidopsis thaliana.
Plant Physiol
107:
895-904
[Abstract]
Monroe JD,
Salminen MD,
Preiss J
(1991)
Nucleotide sequence of a cDNA clone encoding a beta-amylase from Arabidopsis thaliana.
Plant Physiol
97:
1599-1601
Murashige T,
Skoog F
(1962)
A revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiol Plant
15:
473-479
[CrossRef]
Nakamura K,
Ohto M,
Yoshida N,
Nakamura K
(1991)
Sucrose-induced accumulation of
Ohto M-A,
Nakamura-Kito K,
Nakamura K
(1992)
Induction of expression of genes coding for sporamin and
Ougham HJ,
Davis TGE
(1990)
Leaf development in Lolium temulentum. Gradients of RNA complement and plastid and non-plastid transcripts.
Physiol Plant
79:
331-338
[CrossRef]
Ourry A,
Kim TH,
Boucard J
(1994)
Nitrogen reserves mobilization during regrowth of Medicago sativa L.
Plant Physiol
105:
831-837
[Abstract]
Pujadas G,
Ramirex FM,
Valero R,
Palau J
(1996)
Evolution of
Rorat T,
Sadowski J,
Grellet F,
Daussant J,
Delsny M
(1991)
Characterization of cDNA clones for rye endosperm-specific
Sadowski J,
Rorat T,
Cooke R,
Delseny M
(1993)
Nucleotide sequence of a cDNA clone encoding ubiquitous beta-amylase in rye (Secale cereale L.).
Plant Physiol
102:
315-316
[CrossRef][ISI][Medline]
SAS Institute (1989) SAS/STAT Guide for Personal Computer, version
6. SAS Institute, Cary, NC
Sharma R,
Schopfer P
(1987)
Phytochrome-mediated regulation of
Shure M,
Wessler S,
Fedoroff N
(1983)
Molecular identification and isolation of the waxy locus in maize.
Cell
35:
235-242
[CrossRef][ISI][Medline]
Smith D
(1962)
Carbohydrate root reserves in alfalfa, red clover and birdsfoot trefoil under several management schedules.
Crop Sci
2:
75-78
-amylase signature motifs
predicted by the PROSITE program (Bairoch, 1994). Alignment of the
amino acid sequences of -amylase from several plant species (Fig.
1A) shows that the encoded alfalfa
-amylase contains the eight highly conserved motifs identified by
Pujadas et al. (1996). These authors indicated that these motifs define
structural and/or functional elements of -amylases. The predicted
amino acid sequence of MSAB1 is nearly identical with those of white
clover (96%) and soybean (92%). Phylogenetic dendrogram analysis
reveals that MSBA1 is more closely related to white clover and soybean
-amylases than with other plant -amylases (Fig. 1B). The
dendrogram shows that MSBA1 is clustered with -amylases from dicots
and is clearly separated from monocots. The MSBA1 lacks the Gly-rich
repeated motif found in the COOH terminus of rye and barley endosperm
-amylases (Fig. 1A).
View larger version (51K):
[in a new window]
Figure 1.
A, Deduced amino acid sequence of MSBA1 aligned
with sequences of some selected plant -amylases obtained from
GenBank. The multiple alignment was done with the PILEUP program of the
DNA sequence analysis package from Genetics Computer Group. The eight
highly conserved regions of all -amylases identified by Pujadas et
al. (1997) are boxed. B, Phylogenetic tree of plant -amylases.
-Amylase protein sequences of different species were obtained from
GenBank. The protein sequences were aligned using PILEUP. DISTANCE and
GROWTREE programs (Genetics Computer Group) were used to analyze the
evolutionary distances and create the phylogenetic dendrogram.
-Amylase Gene
-amylase gene in the
tetraploid alfalfa genome, genomic DNA was digested with various
restriction enzymes (Fig. 2). Two strong
hybridizing bands were seen in the lanes where DNA was digested with
HindIII, EcoRV, and NdeI. Three
hybridizing bands were obtained for BglII and one strong
band with several weak bands for EcoRI. Because pMSBA1 does
not have NdeI or BglII sites, we expected to
observe one hybridizing band if -amylase were present as a single
gene (except for interruptions by an intron), but instead these enzymes
gave more than one band. We expected two bands from the
HindIII and EcoRV digests, since both have a
single restriction site in the pMSBA1 cDNA. Because of the
hybridization patterns we obtained, it was difficult to tell whether
-amylase was present as a single gene. We then made a probe to only
one of the HindIII fragments from pMSBA1 and reprobed the
Southern blots. We obtained the same hybridization pattern (Fig. 2) as
when the entire cDNA was used as a probe. Our conclusion was that
-amylase was encoded by more than one gene.
-Amylase
-amylase gene was examined in leaves, stems,
and roots of alfalfa and in the roots of sweetclover, white clover, red
clover, and birdsfoot trefoil. Transcripts for -amylase were 100- and 50-fold more abundant in alfalfa roots than in leaves and stems,
respectively (Fig. 3A). The -amylase
transcript was 20-fold more abundant in roots of alfalfa compared with
roots of sweetclover and 50-fold more abundant compared with roots of white clover and red clover (Fig. 3B). The -amylase transcript was
not detected in roots of birdsfoot trefoil.
View larger version (39K):
[in a new window]
Figure 3.
Tissue- and species-specific expression of the
-amylase gene. A, Total RNA from leaves, stems, and roots of
alfalfa. B, Total RNA from roots of alfalfa (ALF), white clover (WC),
sweetclover (SC), red clover (RC), and birdsfoot trefoil (BFT) at the
time of flowering. Northern blots were hybridized with the
32P-labeled pMSBA1 -amylase insert.
-Amylase
Activity, and -Amylase Transcript Levels
-amylase activity, and -amylase gene expression because of the potential role of -amylase in starch degradation. Defoliation reduced root starch concentrations within 7 d after herbage removal, and starch concentrations in roots of these plants remained low for the remainder of the study (Fig.
4A). Because root protein levels also
decline after defoliation, -amylase activity expressed on a protein
basis was similar in roots of defoliated and intact alfalfa plants
(data not shown). However, when expressed on a fresh-weight basis,
-amylase activity declined from d 4 to 24 (Fig. 4B).
View larger version (26K):
[in a new window]
Figure 4.
Changes in starch concentration, -amylase
activity, and -amylase transcript levels as influenced by
defoliation. Greenhouse-grown plants were defoliated at flowering.
Roots were sampled immediately (0 h), at 3, 6, and 12 h, and at 1, 2, 4, 8, 12, 16, 20, 24, and 28 d after defoliation and analyzed
for starch (A) and for -amylase activity (B). In B, U is units. The
LSD is shown at the 5% probability level. C, Total RNA (20 µg) was analyzed by northern analysis using radiolabeled -amylase
cDNA, and the membrane was stripped and reprobed with a
32P-labeled alfalfa 18S ribosomal cDNA. C, Cut (defoliated)
plants; U, uncut, intact control plants. Fr. wt., Fresh weight.
-amylase transcript abundance and its relationship to
defoliation-induced declines in -amylase activity. -Amylase mRNA
abundance declined within 12 h after defoliation, attaining very
low levels on d 2, 4, and 8 (Fig. 4C). Reaccumulation of -amylase
transcript began on d 12 and reached levels similar to that observed in
roots of intact plants by d 28. The -amylase transcript levels did not change in roots of intact plants during this period. The decline in
-amylase transcript levels was more rapid than the decline in
-amylase activity (Fig. 4, B and C). This lag in the loss of
-amylase activity may be due to the relative slow turnover and
stability of the -amylase polypeptide. -Amylase from mustard was
also shown to be relatively stable, with a slow turnover rate (Subbaramaiah and Sharma, 1989).
-Amylase Activity, and mRNA in Cells Grown in
Suc-Rich and Suc-Deprived Medium
-amylase has been
reported in other species (Nakamura et al., 1991; Ohto et al., 1992;
Mita et al., 1995, 1997). We determined how starch concentrations, -amylase activity, and the -amylase transcript levels were
influenced by alfalfa cell cultures grown in B5g medium with or without
Suc. Starch levels declined between 0 and 72 h after inoculation
when cells were grown in B5g medium containing mannitol (Fig.
5B). Starch concentrations increased
during this same time when cells were grown in B5g medium containing
Suc. -Amylase activity did not change significantly in Suc-grown
cells, whereas -amylase activity declined significantly in
mannitol-grown cells at 24 and 48 h (Fig. 5A). The decline in
starch levels in mannitol-grown cells is not associated with elevated
-amylase activity. High levels of -amylase mRNA were maintained
in cells grown in Suc, but -amylase mRNA declined substantially
within 24 h for cells grown in mannitol (Fig. 5C). As observed for
the plant defoliation experiment above, the decline in -amylase
transcript levels was more rapid and substantial than the decline in
-amylase activity, suggesting high stability of the polypeptide.
View larger version (44K):
[in a new window]
Figure 5.
Levels of starch (A), -amylase activity (B),
and -amylase transcript (C) during incubation of alfalfa cell
cultures in Suc-containing and Suc-deprived B5g medium. Cell cultures
were sampled at the time of inoculation (Inoc; 0 h) and 24, 48, and 72 h after cultures were transferred to Suc-free medium, and
then starch, -amylase, and -amylase mRNA were measured. U,
Units.
-Amylase
Activity, and -Amylase Transcript Levels
-amylase activity, and -amylase transcript
abundance. In general, trends in -amylase activity paralleled
changes in starch concentrations. Starch concentrations increased
significantly from September to October in roots of cv Norseman-L and
cv Norseman-O, the two most fall-dormant, winter-hardy germ plasms, and
then, as expected, declined between October and December (Fig.
6A). Starch concentrations also declined
in roots of cv CUF 101-L, the most fall-dormant, winter-hardy cv CUF
101 germ plasm, between September and November (Fig. 6A). In the
remaining populations starch concentrations either remained unchanged
or increased between September and December. -Amylase activity was
measured to determine whether the decline in starch concentration was
associated with high -amylase activity. When expressed on a
dry-weight basis, -amylase activity did not interact with germ plasm
but did increase from 8.9 units/mg in September to 10.3 units/mg in
November and December.
View larger version (68K):
[in a new window]
Figure 6.
Changes in starch concentration, -amylase
activity, and -amylase transcript abundance in roots of alfalfa germ
plasms exhibiting contrasting fall dormancy during winter hardening in
autumn. Roots were sampled from field plots in September, October,
November, and December. Starch levels (A) and -amylase activity (B)
were measured. The LSD is shown at the 5% probability
level. U, Units. C, Total RNA (20 µg) was analyzed by northern
analysis using radiolabeled -amylase cDNA and the membrane was
stripped and reprobed with a 32-P-labeled alfalfa 18S
ribosomal cDNA.
-amylase (Fig. 6B). -Amylase activity declined from September to December in roots of cv CUF 101-L
but did not change in cv CUF 101-O, whereas -amylase activity increased in cv CUF 101-H roots from September to November and then
declined in December. -Amylase activity increased from September to
October in roots of cv Norseman-L and cv Norseman-O and then, like root
starch concentrations, decreased after October. In contrast, -amylase activity decreased from September to October and then increased in November in roots of cv Norseman-H. This consistent association of high -amylase activity with high root starch
concentrations suggests that declines in root starch concentrations
were not caused by higher -amylase activity. Northern analysis
showed that -amylase transcript levels increased from September to
October in all germ plasms except Norseman-H (Fig. 6C). The high
transcript levels for -amylase in September and October declined
markedly by November and December for all germ plasms. Although large
declines in -amylase mRNA occurred between October and December,
-amylase activity remained high in roots of all populations,
suggesting high stability in planta for the -amylase polypeptide.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-amylase cDNA that we
believe encodes the -amylase polypeptide previously described (Boyce and Volenec, 1992b) for several reasons. First, the molecular mass of
the encoded -amylase polypeptide (approximately 55,950 D) is similar
to the 57-kD estimate for the -amylase polypeptide obtained
previously using SDS-PAGE (Boyce and Volenec, 1992b). Second, tissue
distribution of the -amylase transcript and -amylase activities
agree with both being much more abundant in roots than in leaves or
stems. Third, -amylase transcript levels are very abundant in roots
of alfalfa. Lesser amounts were detected in roots of red clover, sweet-
clover, and white clover, and amounts were below our detection limit in
roots of birdsfoot trefoil. -Amylase activity was also found to be
more prevalent in roots of alfalfa than red clover, with virtually no
activity in roots of birdsfoot trefoil (Boyce et al., 1992; Li et al.,
1996). The -amylase polypeptide constitutes 8% of the total soluble
protein fraction in roots (Boyce and Volenec, 1992b). Similarly, we
found large quantities of -amylase mRNA in alfalfa roots, as
indicated by the short exposure time (less than 5 min) needed for
obtaining signals on radiographic films.
-amylase in alfalfa roots remains obscure.
In this study we examined how treatments known to reduce tissue starch
concentrations (defoliation, Suc-deprivation of suspension-cultured
cells, and cold acclimation) influence -amylase activity and
transcript abundance. Taproots of uncut alfalfa plants showed high
starch concentrations, high -amylase activities, and abundance of
the -amylase transcript (Fig. 4). Defoliation reduced root starch
levels but, contrary to our expectations for starch hydrolase, also
resulted in lower -amylase activity and transcript levels. We have
also shown that suspension-cultured cells grown in Suc-rich medium
maintain high starch levels, elevated -amylase activity, and higher
quantities of the -amylase transcript compared with cells deprived
of Suc, which are forced to use starch as a carbon source (Fig. 5).
; Li et al.,
1996), we found that cold acclimation of alfalfa stimulated starch
degradation in autumn in winter-hardy germ plasms but without a
concomitant increase in -amylase activity or transcript level (Fig.
6). Roots of birdsfoot trefoil contain very low -amylase activity
(Li et al., 1996), and the -amylase transcript was not detectable;
yet starch degradation occurs in a similar manner in roots of birdsfoot
trefoil and alfalfa after defoliation (Boyce and Volenec, 1992b) and
during cold acclimation (Li et al., 1996). The amount of -amylase
mRNA in alfalfa, red clover, and birdsfoot trefoil correlates to the
specific activity of total amylase activities observed previously for
these forage legumes (Li et al., 1996). Our data suggest that
-amylase is not a key enzyme in starch degradation in roots of these
forage legumes, because accumulation of the transcript and enzymatic
activity did not coincide with the loss of root starch.
-amylase are found in starch-storing organs
(Doehlert et al., 1982; Yoshida and Nakamura, 1991; Boyce and Volenec,
1992a). We found high -amylase mRNA levels in stems of alfalfa even
though this tissue contains little starch. Wang et al. (1995) isolated
a phloem-specific -amylase from Streptanthus tortuosus
(Brassicaceae). Because monoclonal antibodies made to this -amylase
cross-reacted with -amylase from Arabidopsis, it was suggested that
the major form of Arabidopsis -amylase could be a phloem-specific
enzyme. These authors speculated that the phloem-specific -amylase
may function to hydrolyze maltodextrins in sieve elements to prevent
the accumulation of large polysaccharides that could impede carbon
transport (Wang et al., 1995). Pea epicotyl -amylase was shown to
hydrolyze maltodextrins with few Glc molecules (Lizotte et al., 1990).
Whether alfalfa -amylase has a similar role in the hydrolysis of
maltodextrin in phloem tissue of alfalfa stems is not known.
-amylase is not clear, perhaps signals
that enhance or repress expression of the -amylase gene may lend
some clue as to its function. Sugar-inducible expression of the
-amylase gene is well documented in Arabidopsis and sweet potato
(Nakamura et al., 1991; Ohto et al., 1992; Mita et al., 1995, 1997). In
agreement, cell cultures grown in Suc-free medium reduced -amylase
mRNA, activity, and starch concentrations (Fig. 5). Ironically, roots
of field-grown alfalfa accumulate a significant quantity of soluble
sugars in November and December (Cunningham et al., 1998), and yet
during this period levels of -amylase mRNA are very low compared
with the high mRNA levels in September and October (Fig. 6), when sugar
concentrations are low. Mita et al. (1997) proposed that the gene for
Arabidopsis -amylase is regulated by a combination of both positive
and negative signals that are dependent on the level of sugars.
Southern analysis shows that -amylase is encoded by a small
gene family (Fig. 2), members of which may be regulated by different
sugars or sugar concentrations. Three -amylase isozymes reported
previously from alfalfa taproots (Doehlert et al., 1982; Habben and
Volenec, 1991) may each be encoded by a different member of the
-amylase gene family. In contrast, -amylase from Arabidopsis and
maize is encoded by a single gene (Mita et al., 1995; Wang et al.,
1997).
View larger version (27K):
[in a new window]
Figure 2.
Southern analysis of the -amylase gene. Genomic
DNA (10 µg) was digested separately with restriction enzymes, as
indicated in each lane, and analyzed by hybridization with the
radiolabeled -amylase probe.
-amylase
is not clear, it has been proposed that -amylase may function as a
VSP, providing the nitrogen needed for shoot regrowth (Hendershot and
Volenec, 1993b; Li et al., 1996). This is an attractive hypothesis
because -amylase conforms to the definition of VSPs based on the
perceived physiological function described by Cyr and Bewley (1990).
VSPs are preferentially synthesized in storage organs and exhibit
depletion during reactivation of meristems, and their abundance greatly
exceeds that of other proteins in perenniating organs. -Amylase
conforms to these definitions of a VSP because it is more abundant in
alfalfa roots than in stems and leaves. Defoliation reactivates
meristems on crowns for shoot regrowth and substantially reduces
-amylase activity and -amylase transcripts.
-amylase
(Boyce and Volenec, 1992a). Defoliation reduces nitrogenase activity
and nitrogen fixation (Vance et al., 1979). Alfalfa root -amylase
shows patterns of activity similar to three abundant VSPs we have
characterized from alfalfa roots (Cunningham and Volenec, 1996). These
four polypeptides (including -amylase) accumulate in alfalfa
taproots in early autumn and disappear in spring, when shoot growth
resumes, and during shoot regrowth when plants are defoliated. As shown
by 15N labeling, significant movement of nitrogen
occurs from this VSP-enriched protein pool to shoots after defoliation
(Avice et al., 1996; Barber et al., 1996). Our data support the view
that -amylase from alfalfa roots functions as a VSP.
-amylase gene by winter hardening and defoliation stress in alfalfa roots. Further work will concentrate on enhancing our
understanding of the function of -amylase using antisense DNA
transformation studies. Work is also under way to clone and characterize the cDNAs for the three alfalfa taproot VSPs that we
described previously (Cunningham and Volenec, 1996). We hope that
understanding the structural and biological features of these VSPs will
reveal the reasons that -amylase serves a similar role in the roots
of this species.
1
The work was supported by U.S. Department of
Agriculture grant no. 96-35100-3141.
FOOTNOTES
*
Corresponding author; e-mail jvolenec{at}purdue.edu; fax
1-765-496-2926.
ABBREVIATIONS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-Amylase from taproots of alfalfa.
Phytochemistry
31:
427-431
[CrossRef]-amylase.
Plant Physiol
94:
1033-1039
-amylase occurs concomitant with the accumulation of starch and sporamin in leaf-petiole cuttings of sweet potato.
Plant Physiol
96:
902-909
-amylase by polygalacturonic acid in leaves of sweet potato.
Plant Physiol
99:
422-427
-amylase: patterns of variation and conservation in subfamily sequences in relation to parsimony mechanisms.
Proteins Structure Function Genet
25:
456-472
[CrossRef]-amylase and analysis of -amylase deficiency in rye mutant lines.
Theor Appl Genet
83:
257-263
[CrossRef]-amylase mRNA in mustard (Sinapsis alba L.) cotyledons.
Planta
171:
313-320
-Amylase from Alfalfa Taproots