Evolutionary significance of differential regulation at the wx locus of rice

The waxy ( wx ) locus in rice, which specifies a major starch granule-bound protein
called Wx protein, is genetically well characterized. In addition to wx, at least two
nonwaxy alleles at the wx locus regulate the amount of the gene product as well
as amylose content (AC). The present study was carried out to learn to what
extent allelic differentiation at the wx locus is important for the diversity in AC
observed among nonwaxy cultivars. Among the naturally occurring variants tested,
AC was determined mainly by allelic changes at the wx locus. In addition, the
amount of the gene product was affected by temperature, modifiers, and gene
dosage in the same way AC was affected. These effects imply that AC is a quantitative
trait in segregating populations as is often reported in rice, even though it
is controlled simply by the amount of Wx protein. Allelic differentiation at the wx

locus is discussed in relation to the evolutionary significance of gene regulation.
Amylose content (AC), a major determinant of eating quality, varies greatly among rice
cultivars. The diverse AC phenotypes seem to have arisen during domestication, since
the wild progenitor shows no such tendency. The waxy ( wx ) locus in rice affects the
type of starch produced in the endosperm tissue. The simple inheritance contrasts
markedly with the polygenic nature of AC observed among nonwaxy (or nonglutinous)
cultivars. Accumulated evidence has revealed that higher organisms contain substantial
genetic factors regulating gene expression. Recently, we proposed that there are
two nonwaxy alleles at the wx locus in rice and that they regulate the quantitative level
of the gene product differently, suggesting the presence of regulatory site(s) (Sano
1984). This presents a unique opportunity to examine the evolutionary change of
regulatory elements that could be selected for. We present evidence here that differential
expression at the wx locus greatly contributes to the continuous variation in AC
observed in rice cultivars.
Determinants of amylose content
The wx locus specifies a 60-kDa major protein tightly bound to the starch granules
(Sano 1984). The wx locus is the structural gene for the 60-kDa protein called Wx
11
protein, since a wx mutant (75-1) produced an altered form of this protein. The amount
of the gene product increased in triploid endosperms through dosage effects in the same
manner in which AC had been shown to increase. The wx locus in maize has been well
characterized by biochemical and molecular techniques, and the antigenic relatedness
of the Wx proteins in maize and rice (Okagaki and Wessler 1988) and their DNA
sequence similarity (Hirano and Sano, unpubl. data) imply that both loci encode uridine
diphosphoglucose-glucose starch glycosyl transferase. The assumption that the amount
of Wx protein also causes varying degrees of AC among nonwaxy cultivars arose from
the observation that two electrophoretically distinct alleles, Wx a and Wx b , at the wx
locus control different amounts of the gene product as well as AC (Sano 1984). Wx a
produces about 10 times as much gene product as Wx b , and the 2 alleles act additively
in triploid endosperms. A positive correlation between the amount of Wx protein and
AC (Sano et al 1985a) suggests that AC might be a good indicator of enzymatic activity
of the gene product. Enzymatic activity is determined by the amount of gene product,
suggesting the presence of cis-acting regulatory site(s) near or within the structural
gene.
Allelic differentiation at the wx locus
Polyacrylamide gel analysis in extracts from starch granules readily distinguished
most alleles as Wx a or Wx b , although some showed intermediate amounts of Wx protein
and amylose, and their allelic states remain unknown (Sano et al 1985a). The
quantitative differences might have been caused by a modifier that is independent of
wx or the Wx a and Wx b alleles at the wx locus. To clarify their allelic states, alleles at
the wx locus were introduced into a waxy line (T65 wx, a near-isogenic line of Taichung
65 with wx ) by successive backcrosses from 17 nonwaxy lines including different taxa.
The alleles introduced into the same genetic background showed amounts of Wx
protein and amylose similar to those found in the original lines (Fig. 1, Table 1). The
near-isogenic lines with the alleles from cultivars that showed intermediate amounts
of Wx protein and amylose also gave the same tendency. This confirmed that the alleles
1. Comparisons of starch granule-bound Wx proteins among 5 near-isogenic lines (A-E) of Taichung 65
with alien alleles at the wx locus, and their donor parents (H-L). A and H = W025 ( O. glaberrima ), B and
I = Patpaku (indica type of O. sativa ), C and J = W593 ( O. rufipogon ). D and K = Macan Coranglan (javanica
type of O. sativa ), E and L = Nagaewase (japonica type of O. sativa ), F = Taichung 65 with Wx b (japonica
type of O. sativa ), G = near-isogenic line of Taichung 65 with wx from Kinoshitamochi, the recurrent parent.
12 Sano et al
Table 1. Four types of nonwaxy alleles found in 17 accessions of Oryza spp. a
Wx protein
Amylose
Type Relative 2-D content d Distributioe n
amount b pattern c
I
(1)
II
Ill
IV
High
(9.4-11.3)
High
(9.7-10.6)
Low
(0.9-1.0)
Intermediate
(2.2-3.6)
A
B
A
A
High
(25.7-29.1)
High
(24.8-28.1)
Low
(15.2-15.8)
Intermediate
(20.3-22.5)
O. sativa indica (3), O. rufipogon
(4), O. longistaminata
O. glaberrima (3), O. barthii (1)
O. sativa japonica (2)
O. sativa japonica (1), O. sativa
javanica (2)
a Gene expressions were analyzed after the alleles at the wx locus were introduced into a waxy line by successive
backcrosslng. T65 wx (a near-isogenic line of Taichung 65 with wx ) was used as the recurrent parent The
backcross generations differed from BC 3 to BC 11 depending on the line. b The amount in Norin 8 was regarded
as unity. Numbers in parentheses are ranges in percent. c A and B correspond to A and B in Figure 3, respectively,
Types I and II were reported as Wx a and Type III as Wx b (Sano 1984). d Numbers in parentheses are ranges in
percent. e Number of accessions in parentheses.
that specify the intermediate amounts are neither Wx a nor Wx b . This is supported by the
observation that intermediate AC is controlled by a major gene (Kumar and Khush
1987). These results strongly suggest that the major gene is the allelic change at the wx
locus itself.
Two-dimensional electrophoresis revealed heterogeneity of the gene product. Four
polypeptides in soluble proteins were specific to nonwaxy lines, since those polypeptides
disappeared in waxy lines (Fig. 2). The four polypeptides were detected in all
nonwaxy lines examined. However, one polypeptide was not detected in the starch
granule-bound proteins. In addition, the position of the missing polypeptide was
different between lines with alleles from O. glaberrima and O. sativa (Fig. 3). The
heterogeneity of the gene product appeared to result from modifications after transcription,
since a wx mutant (75-1) showed a simultaneous change in those polypeptides.
Based on these results, four types of alleles were recognized among nonwaxy
accessions (Table 1).
Induced mutants affecting gene expression
In naturally occurring variants it is difficult to interpret the regulatory function without
the use of near-isogenic lines, because their genetic backgrounds may lead to misinterpretation,
and their evolutionary origins are obscure. Comparing induced mutants
of maize and rice, Amano (1981) found that leaky (or low-AC) mutants were more
frequently induced in rice. Induced mutants obtained from Norin 8 were distinguished
by their endosperm appearance, and their phenotypic changes were controlled by a
recessive gene. Allelism tests were conducted to examine if the mutant genes were
allelic to wx. The 12 wx mutant alleles were classified into 4 types according to the
Differential regulation at the wx locus of rice 13
2. Heterogeneity of Wx proteins (spots 1–4) revealed by 2-dimensional polyacrylamide electrophoresis in
total proteins solubilized from endosperm with Tris-HCl buffer (pH 6.8). A = Taichung 65 with Wx b , B =
near-isogenic line of Taichung 65 with Wx a from O. glaberrima. IEF and SDS show isoelectric focusing
dimension and sodium dodecyl sulfate dimension, respectively.
amounts of Wx protein and amylose (Table 2). This contrasts with the fact that all waxy
cultivars tested showed no Wx protein and almost no amylose (Sano 1984). On the
other hand, none of the five low-AC mutants were allelic to wx, and all showed different
segregation ratios when crossed with T65 wx. The mutant alleles were independent of
each other and had different effects on endosperm appearance. They were classified
into four types according to their phenotypic differences and segregation patterns
(Table 2). The low-AC mutant gene generally showed no gene dosage effect in the F 2
(Okuno et al 1983, Sano et al 1985a), suggesting that the gene product acts as a transacting
regulatory factor. In addition, the manner of trans-action markedly differed
between plants with Wx a and those with Wx b , since a mutant allele reduced AC only in
plants with Wx b (Kikuchi and Kinoshita 1987).
14 Sano et al
3. Starch granule-bound Wx proteins as
revealed by 2-dimensional polyacrylamide
electrophoresis. Of the 4 polypeptides
(spots 1–4) detected in total proteins as
shown in Figure 2, only 3 were found
(open triangle indicates no spot). A =
Taichung 65 with Wx b , B = near-isogenic
line of Taichung 65 with Wx a from O.
glaherrima.
Table 2. Classification of induced mutants affecting AC in rice endosperms according to
gene expression at the wx IOCUS. a
Amylose Relative amount
content of Wx protein Ratio
F 2
Type segregation Mutants
(%) (b) in cross
(a) with T65wx
wx mutants
I 0.0
II
III
0.3-0.6
0.0
IV 1.2
Low-AC mutants
I 7.2-7.4
II 5.1
III 4.6
IV 3.4
Controls
Norin 8 15.6
T65 wx 0.0
0.00
0.61-0.77
0.19-0.56
0.09
0.26-0.27
0.26
0.70
0.27
1.00
0.0
0.01-0.004
0.00
0.13
0.27-0.28
0.20
0.07
0.13
0.16
No segregation
No segregation
No segregation
No segregation
9:3:4
3:1
9:3:4
9:7
3:1
No segregation
73-1, 74-4, 74-7
74-8, 75-1
74-1, 75-5, 76-1,
76-2, 76-5, 74-2
74-2
74-5, 74-6
74-9
75-2
76-3
a The mutant gene in 76-3 is allelic to du-2.
Differential regulation at the wx locus of rice 15
(a/b)
–
–
Altered gene expression
As mentioned, alleles at the wx locus showed a dosage effect, with continuous variation
of AC in segregating populations. The dosage effect affected AC in different ways
according to the cross examined, which could be explained from allelic differences as
well as the curvilinear regression observed between the amounts of Wx protein and
amylose (Sano 1984, 1985). Another factor affecting AC is temperature. Lower
temperature during grain development increases AC (Cruz et al 1989, Takeda and
Sasaki 1988). Temperature insensitivity has great value for breeding in a region where
low temperatures reduce grain quality because of the increment of AC. The amount of
Wx protein was shown to increase with low temperature in a japonica type having Wx b ,
suggesting that the AC increment is caused by an enhanced level of the gene product
responding to low temperature (Sano et al 1985b). Temperature response was examined
in the near-isogenic line with Wx a and in the mutant line (75-1) with a partially
inactivated form that was induced from Norin 8 with Wx b . The line with Wx a showed
no temperature response, while in 75-1 the amount of Wx protein increased with low
temperature (Fig. 4). These results strongly suggest that temperature response is also
controlled by allelic changes at the wx locus but is independent of an inactivation of the
gene product. The results also support the assumption that AC is controlled through
changes in the amount of gene product, especially by allelic differentiation, even when
the temperature effect is considered (Fig. 4).
Diversification in Asian cultivars
A number of accessions, including wild and cultivated rices, were examined for level
of Wx protein in the endosperm starch. The results showed that the allelic states of the
Relative amount of Wx protein
Amylose content (%)
4. Effects of low temperature on amounts of Wx protein and amylose in 4 rice lines carrying different alleles
at wx locus. L = 20 °C, N = 26 °C. A = near-isogenic line of Taichung 65 with Wx a from Patpaku, B =
Taichung 65 with Wx ,b C = wx mutant (75-1, induced from Norin 8) showing 62-kDa protein that seems
partially inactivated, D = Norin 8 with Wx b . Amount of Wx protein in Norm 8 (26 °C) was taken as unity.
16 Sano et al
near-isogenic lines with different alleles could be estimated from the amount of Wx
protein, since few changes from the original lines were detected, and their alleles could
be classified into three states: Wx a , Wx b , and the intermediate. The distribution of these
alleles among rice taxa with the AA genome clearly showed that only Asian cultivars
have great diversity, since African cultivars and all wild taxa have only the Wx a allele
(Sano et al 1986). In addition, the frequency of the three alleles was markedly different
among three ecogeographical races: Wx a and Wx b were predominant in indica and
japonica types, respectively, whereas the javanica type was heterogeneous. This
strongly suggests that Wx b emerged from Wx a as a mutant with reduced level of gene
product, and that the frequency of the allele increased during evolution of the japonica
type.
Naturally occurring nonwaxy alleles fell into three major types —Wx a , Wx b , and
intermediate-in terms of the amount of gene product; however, there might be
intergrades within each allele, since near-isogenic lines with the same allele differed
slightly in amount of gene product as well as in AC (Table 1).
Evolution at the wx locus
There is little evidence that regulatory changes of gene expression relate to phenotypic
changes. The present results provide strong evidence that differential regulation at the
wx locus plays a significant role in differential AC among diverse phenotypes, which
affects grain quality. The wx locus in rice is not essential for growth, and defective
mutants can survive. However, wx mutant alleles might be slightly deleterious, since
homozygotes are rarer in introgressed wild populations than expected (Oka and Chang
1961). This suggests that the wx allele tends to disappear in wild populations by natural
selection. Similar selection might act against the mutant allele with reduced expression,
since no other taxa except O. sativa contain divergent phenotypes, even though
wx alleles can occur by mutation. An attempt to find a waxy line in African rice ( O.
glaberrima ) was made in vain; however, a wx mutant was induced with ethyl
methanesulfonate treatment. The mutant showed a similar property in the starch
granules (Uematsu and Yabuno 1988) and carried wx at the same chromosomal
location as in O. sativa (Sano 1989), suggesting that African farmers had never selected
wx mutants for their use regardless of their potential. Waxy O. sativa cultivars often
have an independent origin, because intragenic recombinations occur among them.
This clearly shows that farmers have selected wx mutants repeatedly during domestication.
The frequency of intragenic recombination varied according to cross, and the
maximum frequency of revertant pollen exceeded 0.5 × l0 (Li et al 1968). This
suggests that the mutated sites responsible for the waxy phenotype are not restricted to
a limited area of the DNA sequence.
The distribution of Wx b is restricted to East Asian countries where waxy cultivars
prevail. Most Japanese modem cultivars carry Wx b . Allelic states were examined in
traditional cultivars from southern Japan in addition to weedy forms from Japan and
Korea (Table 3). The results suggest that fixation of Wx b took place recently. Moreover,
Differential regulation at the wx locus of rlce 17
–3
Table 3. Distribution of nonwaxy alleles in Japanese rice cultivars and weedy
forms.
Accessions Distribution (no.)
Source (no.)
Wx a Wx b Intermediate
Modern cultivars
Traditional cultivars
from Kyushu
from Okinawa
Weedy forms
25
17
20
14
0
0
8
9
25
6
8
3
0
11
4
2
Wx a itself appears to have no adverse effects, since weedy forms frequently carry it.
Intermediate alleles were also frequently detected in cultivars from Nepal (4 of 26) and
Manipul, India (11 of 24), where no Wx b has been detected. This suggests that the intermediate
allele might have emerged directly from Wx a . However, it is difficult to
speculate whether Wx b and the intermediate alleles have different origins. Recently,
opaque endosperm with about 10% AC was found in an indica from Nepal (Heu and
Kim 1989). Opaque endosperm was controlled by an allele at the wx locus. In the
present study, wx mutants induced from Wx b always showed no or only a trace of
amylose. The opaque mutant might be a leaky mutant but might have a considerable
amount of amylose if it has mutated from Wx a , which produces higher levels of gene
product and amylose than Wx b .
Importance of differential regulation
New functional genes seldom emerge in the evolutionary process. Microevolution in
crops proceeds with mutation, recombination (or hybridization), and selection. The
diversity in gene expression at the wx locus observed only in Asian cultivars is regarded
as the result of utilization of rare mutants by farmers; otherwise, such mutants are
mostly doomed to extinction. In addition to allelic diversity, fluctuations in gene
expression by temperature, modifiers, and dosage effects, if any, tend to make AC a
quantitative trait, similar to most agronomically important traits. Nonfunctional wx
alleles produce only a waxy phenotype, which almost lacks amylose. On the contrary,
the regulatory changes result in various mutants with quantitatively different levels of
gene expression but without losing the gene product entirely. Although the wx locus
is not essential in rice, agronomically important traits like yield and flowering time may
be controlled by essential genes whose dysfunction makes a plant nonviable. In such
a case, only slightly deleterious mutants may be allowed to occur and to be maintained
within a population. Mutants with modified gene expression but without the entire loss
of gene product are expected to be regulatory mutants in nature. Traits like yield and
flowering time may involve more than one basic gene, so regulation becomes more
complex than with the wx locus. Such an example has already been reported in a
continuous pattern of anthocyanin pigmentation, which is controlled by an allelic
18 Sano et al
series at three independent loci in addition to tissue-specific regulatory elements
(Takahashi 1957). To detect major factors from polygenic traits, restriction fragment
length polymorphism analysis will be used. More studies on gene regulation in
quantitative traits are also needed for understanding the polygenic nature of agronomically
important traits and for manipulation of the gene system.