ALL ABOUT DWARFING GENES IN WHEAT

BY:SONAM MEHTA

M.Sc. BIOTECHNOLOGY

GURU NANAK GIRLS COLLEGE

LUDHIANA

Nomenclature

Rht-B1a ( Formerly rht1): Exhibits wild type stature.

Rht-B1b ( Formerly Rht1): Exhibits semi-dwarf stature.

Rht-D1a ( Formerly rht2): Exhibits wild type stature.

Rht-D1b (Formerly Rht2) :Exhibits semi-dwarf stature.

rht8 : Exhibits wild type stature.

Rht8 : Exhibits semi-dwarf stature.

Figure .:Development of plant height from the soil surface to the top ligule. plants at the

7th week in AS(autumn sown), the yellow line shows the site of the top ligule in each

genotype and the two parents.

Reduced height in cereals is often associated with increases in yield due to a reduced risk

of lodging, increase in partitioning of assimilates to the grain (Evans, 1993), and more

fertile florets per spikelet (Brooking and Kirby, 1981). Wheat is the most important cereal

grain crop in the world.It is the staple crop for about 35% of the human population and

also known as “king” of the cereals (Laghari et al 2010).It is a crop that has profound

social and economic importance across countries. It is the principal cereal grain crop used

for food consumption in the United States and most parts of the world. The leading

producers being China, India, Turkey, Pakistan, and Argentina; INDIA is second largest

producer of wheat in the world .World production of wheat in 2001 was 583.9 million

metric tons, occurring on 219.5 million acres. World wheat consumption in that period

was 590.6 million tons. The consumption of wheat has been increasing with the

increasing population and thus its production need to be increased. It is estimated that

over the next decade, grain production must increase by 15% to meet the global demand

and consumption of wheat as a result of a growing human population (Edgerton, 2009).

Improving and achieving yield stability remains a daunting challenge.One strategy to

meet this challenge is to increase wheat productivity by optimizing plant architecture

(defined by tillering, stature, and leaf and ear morphology). Plant architecture is of major

agronomic importance as it determines the adaptability of a plant to cultivation, harvest

index, and potential grain yield (Reinhardt and Kuhlemeier, 2002).

A decisive component of plant architecture is stature, mainly determined by stem

elongation. Wheat (Triticum aestivum L.) is an annual crop with round, hollow, and

jointed culms (stems). There are usually five elongated internodes in fully grown culms,

with each internode progressively longer towards the ear. The internode elongation,

which determines final plant height, is regulated by genes involved in brassinosteroid

(BR) and gibberellin (GA) biosynthetic or signalling pathways.Semi-dwarfing genes in

wheat made a significant contribution to the ‘Green revolution’ in the 1960s .Twenty-one

reduced height genes in wheat, Rht1 to Rht21, have been described till .Among them,

four genes, originally named Rht1, Rht3, Rht2 and Rht10, were re-designated as Rht-

B1b, Rht-B1c, Rht-D1b and Rht-D1c, respectively and these were shown to be alleles at

two loci. Semidwarfing genes led to the higher yields due to improved lodging resistance

and the resulting ability to tolerate higher rates of inorganic nitrogen-based fertilizer

(Gale and Youssefian, 1985). The decrease in stem stature resulted in an increase in

assimilate partitioning to developing ears, enabling greater floret survival at anthesis and

increased grain numbers per ear (Youssefian et al., 1992).-

Green revolution

The term “Green Revolution” refers to the huge increases in grain yields after the 1960s,

resulting from the introduction of new varieties of wheat and rice, particularly for use in

the developing world. This development was a major factor in maintaining per capita

food supplies worldwide in the late 20th Century despite a doubling in the world

population during this time (Evans, 1998) and was recognised by the award in 1978 of

the Nobel Peace prize to Norman Borlaug of the International Maize and Wheat

Improvement Center (CIMMYT). Borlaug developed high yielding wheat varieties

suitable for growing in sub-tropical and tropical climates. The higher grain yields were

obtained in part through increased use of fertilizers and pesticides. However, the heavier

grain caused the plants to become unstable and prone to lodging (falling over) in high

winds and rain. Borlaug introduced dwarfing genes into wheat giving the plants a

stronger, shorter stem that resisted lodging.The advantages of using dwarfing genes with

high-yielding varieties was soon recognized and most commercial wheat varieties contain

such genes, in temperate as well as in sub-tropical regions. An additional benefit from

these genes has been an increase in grain yield through an improvement in the ‘harvest

index’ (the proportion of plant weight in the grain). This means that a greater proportion

of the products of photosynthesis accumulates in the grains rather than in the leaves.

Modern wheat varieties have a harvest index of over 50%, with a sharp increase since the

introduction of the dwarfing genes (Evans, 1998).

The Dwarfing Genes of wheat

The dwarfing genes in wheat are classified into two categories, GA-responsive (GAR)

and GA-insensitive (GAI), reflecting the relative magnitude of their responses to

application of exogenous GA. GA-responsive dwarfing genes show significantly

enhanced growth response to exogenous GAs (probably have mutations in GA

biosynthesis pathway) while GA-insensitive dwarfing genes show very little response to

exogenous GAs (probably have mutations in GA signaling pathway, such as Rht-D1b and

Rht-B1b) . This classification is conducted at the seedling stage, for example, based on

the response of coleoptile length or the first seedling leaf elongation rate to exogenous

GAs .The genes associated with a semi-dwarf growth habit in wheat are known as

Reduced height (Rht) genes and many of them are dominant or semi-dominant, indicating

that they actively inhibit growth through a so-called gain-of-function mutation. Twenty-

one reduced height genes in wheat, Rht1 to Rht21, have been described. Among them,

four genes, originally named Rht1, Rht3, Rht2 and Rht10, were re-designated as Rht-

B1b, Rht-B1c, Rht-D1b and Rht-D1c, respectively, when they were shown to be alleles at

two loci.

Rht-B1b (Rht 1) and Rht-D1b (Rht 2)

The Rht-B1b (Rht1) and Rht-D1b (Rht2) semi-dwarfing genes were introduced into

commercial wheat cultivars from the Japanese variety Norin10 in the 1960s as part of

wheat improvement programs in the USA and at CIMMYT, Mexico. A reduction in plant

height improved lodging resistance and partitioning of assimilates to the developing grain

(Evans 1993).

Figure. : Rht B1b sequence in Triticum aestivum (from NCBI).

Figure3. : The conserved sequence of Rht-B1 ( Pearce et.al, 2011).

Figure4. : Sequence of Rht D1gene in Triticum aestivum (from NCBI).

The large increases in yield that followed the introduction of these dwarfing genes led to

widespread adoption of the dwarfing genes throughout the world (Gale et al. 1985).

Recently, the homoeologous genes Rht-B1b and RhtD1b were isolated from wheat (Peng

et al. 1999). They are orthologous to the Arabidopsis GAI gene, a de-repressible

modulator of gibberellic acid (GA) response (Peng et al. 1997). Both the Rht-B1b and

Rht-D1b mutations are associated with a single base-pair change leading to a TAG stop

codon shortly after the start of translation (Peng et al. 1999). These mutations reduce the

plant’s ability to respond to GA, so that exogenous application of this hormone does not

restore wild-type plant height. Hence the presence of these dwarfing genes can be

determined by testing seedlings for the lack of responsiveness to GA (Gale and Gregory

1977; Richards 1992). Although relatively easy, this test is time-consuming, not always

reliable, and does not discriminate between Rht-B1b and Rht-D1b. These limitations can

be overcome by using molecular markers for these dwarfing genes. We developed PCR-

based markers aimed at discriminating between mutant (dwarf) Rht-B1b and Rht-D1b

and their wild-type (tall) alleles.

Rht8

The Reduced height 8 (Rht8) semi-dwarfing gene is one of the few, together with the

Green Revolution genes, to reduce stature of wheat (Triticum aestivum L.), and improve

lodging resistance, without compromising grain yield. Rht8 is widely used in dry

environments such as Mediterranean countries where it increases plant adaptability. With

recent climate change, its use could become increasingly important even in more northern

latitudes. In the present study, the characterization of Rht8 was furthered. Morphological

analyses show that the semi-dwarf phenotype of Rht8 lines is due to shorter internodal

segments along the wheat culm, achieved through reduced cell elongation. Physiological

experiments show that the reduced cell elongation is not due to defective gibberellin

biosynthesis or signalling, but possibly to a reduced sensitivity to brassinosteroids. Using

a fine-resolution mapping approach and screening 3104 F2 individuals of a newly

developed mapping population, the Rht8 genetic interval was reduced from 20.5 cM to

1.29 cM. Comparative genomics with model genomes confined the Rht8 syntenic

intervals to 3.3 Mb of the short arm of rice chromosome 4, and to 2 Mb of Brachypodium

distachyon chromosome 5. Rht8 appears to be of importance to South European wheats

as alternative giberellic acid (GA)-insensitive dwarfing genes do not appear to be adapted

to this environment.The very successful semi-dwarf varieties bred by CIMMYT, Mexico,

for distribution worldwide have been thought to carry Rht8 combined with GA-

insensitive dwarfing genes. Additional height reduction would have been obtained from

pleiotropic effects of the photoperiod-responsegene Ppd1 that is essential to the

adaptability of varieties bred for growing under short winter days in tropical and sub-

tropical areas. The microsatellite analysis showed that CIMMYT wheats lack Rht8 and

carry a WMS 261 allelic variant of 165 bp that has been associated with promoting

height. This presumably has adaptive significance in partly counteracting the effects of

other dwarfing genes and preventing the plants being too short. Most UK, German and

French wheats carry an allelic variant at the WMS 261 locus with 174 bp. This could be

selected because of linkage with the recessive photoperiod-sensitive ppd1 allele that is

though to off eradaptive significance northern European wheats.The diculty in

recognising Rht8 in varieties has led to many claims concerning the distribution of the

gene worldwide and its potential benefits to breeding programmes. These claims are

difficult to substantiate. Rht8 has been reported to be present in Chinese and CIMMYT

varieties (Mishra and Kushwaha 1995) and to enhance the yield of these wheats. In

reality the only definitivestudies on the effect of Rht8 involve the use of precise

geneticstocks derived from varieties like ‘Mara’ from Italy and ‘Sava’ from Yugoslavia

where both pedigree analysis and observations on defined aneuploid stocks confirm that

Rht8 must be present on the 2D chromosome.The data from thesestudies are restricted to

both a limited range of varieties and limited array of environments. These studies suggest

that Rht8 reduces height by around 8—10 cm in the UK (Worland and Law 1986;

Worland et al. 1988a,b), 5 cm in mid Germany (Worland et al. 1992) and 5—7 cm in

Yugoslavia (Worland et al. 1988a, b, 1990). In all cited examples, data were obtained on

single-chromosome recombinant lines between chromosomes 2D of ‘Mara’ (Rht8) and

‘Cappelle-Desprez’ (rht8) in a homozygous ‘Cappelle-Desprez’ background. Few

significant additive genotypic e ects of Rht8 were detected on other agronomicff

characters. Interactions were detected between Rht8 and Ppd1 for spikelet numbers, grain

size and ear yield (Worland et al. 1988a, b). No interactive environmental effects were

detected for Rht8 when similar lines were tested in England and Yugoslavia. These

results suggest that at least in Europe, within the varietal background, year and

environment limitations of the trials Rht8 could be used to reduce height without adverse

effect on plant yield. Recently a microsatellite marker, WMS 261, has been identified that

shows very restricted recombination with Rht8 (Korzun et al. 1998). Three main allelic

variants of 165 bp, 174 bp and 192 bp have been detected at the WMS 261 locus on the

short arm of chromosome 2D. The 165-bp variant was found to be diagnostic for the

CIMMYT variety ‘Ciano 67’, the 174-bp variant diagnostic for ‘Cappelle Desprez’, the

tall control variety in experiments determining the pleiotropice ects of Rht8, and theff

192-bp variant diagnostic for the Italian variety ‘Mara’, the donor of Rht8. Genetic

analysis of single-chromosome recombinant lines developed in a ‘Cappelle-Desprez’

background between 2D chromosomes of ‘Cappelle-Desprez’ and ‘Ciano67’ shows a

significant height increase of around 3—4 cm associated with the WMS 261 165-bp allele

compared to the WMS 261 174-bp allele. This 3- to 4-cm increase is in addition to the5—

10 cm of the WMS 261 192-bp allele versus WMS 261 174-bp allele comparison. It is

anticipated that the very close linkage of WMS 261 to Rht8 will permit the use of the

microsatellite as a marker for determining the distribution of Rht8 in international

breeding programmes and to demonstrate how Rht8 has been transmitted from

initial.crosses involving the source variety ‘Akakomugi’. In the experiments described

here over 100 varieties have been screened for allelic variants at the WMS261 locus.The

varieties were chosen to include key varieties in the pedigrees of modern varieties that are

thought to carry Rht8, varieties from diverse international breeding programmes and

check varieties that have been shown by cytological observation to carry Rht8 and are

therefore able to be used to verify that recombination has not occurred between WMS

261 and Rht8 and thus verify conclusions drawn from the marker association.

Worldwide distribution of WMS 261 allelic variants

The dwarfing genes credited with playing major roles in improving wheat yields (Rht-

B1b, Rht-B1d, Rht-D1b, Rht8, Ppd1) all seem to have either originated in Japan or to

have been incorporated into early Japanese varieties before spreading into worldwide

breeding programmes. ‘Akakomugi’, the source variety for Rht8, carries the diagnostic

WMS 261-192-bp microsatellite. Of 5 other Japanese varieties tested, 2 modern varieties

‘ChikushiKomugi’ (‘Norin 121’) and ‘Fakuho-Komugi’ (‘Norin 124’)bothcarrythe Rht8

allele,as does‘Haya-Komugi’, an old land-race and a parent of another important Japanese

dwarfing source variety ‘Saitama 27’. ‘Saitama 27’ itself has not obtained the diagnostic

Rht8 allele from ‘Haya-Komugi’ but carries the WMS 261165-bp allele. The third

important Japanese dwarfing gene source variety, ‘Norin 10’, carries the WMS 261174-

bpallele. This indicate that even in the early decades of the twentieth century all three

major WMS 261 alleles were segregating in Japanese wheats.

Rht12

Rht12 has been classified as a GA-responsive dwarfing gene , but its role, if any, in GA

biosynthesis or signalling remains unknown. Rht12 is located on chromosome 5AL,

linked to Xgwm291 at a distance of 5.4 cM. , it was found that the effects of the dwarf

gene. Rht8 which had been considered as ‘GA-sensitive’ was possibly not due to the

defective gibberellin biosynthesis or signalling, but possibly to a reduced sensitivity to

brassinosteroids. Rht12, a dominant dwarfing gene from the gamma ray-induced mutant

Karcagi 522M7K of winter wheat (here referred to as Karkagi-12), has been classified as

a GA-responsive dwarf gene .Rht12 significantly decrease stem length (43%,48% for

peduncle) and leaf length (25%,30% for flag leaf), while the thickness of the internode

walls and width of the leaves were increased. Additionally, the Rht12 dwarf lines showed

very dark green leaves compared to tall lines. Rht12 significantly decreased plant height,

by around 40%, while seedling vigour, coleoptile length and root traits at the seedling

stage were not affected adversely. Rht12 lines had significantly increased floret fertility

and grain number and achieved a higher harvest index (due to the lower plant biomass)

than the tall genotypes. However, Rht12 extended the duration of the spike development

phase, especially the duration from sowing to double ridge, and delayed anthesis date by

around 5 days. Even the dominant Vrn-B1 allele could not compensate for these effects

on phenological development, which may hamper the direct utilization of Rht12 in wheat

breeding. Another negative effect of Rht12 on yield components was that grain size was

reduced significantly. Similarly, other studies have found that Rht12 had a substantial

effect on reducing plant height without altering early vigour and significantly increased

spikelet fertility, harvest index, and lodging resistance but these were usually

accompanied by delayed ear emergence and reduced grain weight.Although Rht12 has

been classified as a GA-responsive dwarfing gene a comprehensive understanding on the

response of Rht12 to exogenous GAs is lacking. Thus, the role of Rht12, if any, in GA

biosynthesis or signaling is still unclear. Moreover, it has been recently found that the

effects of the dwarfing gene Rht8, which had been considered as ‘GA-responsive’, was

possibly not due to defective GA metabolism or signaling because the wild type and Rht8

lines responded with a very similar increase in final plant height (15% and 13%,

respectively; P,0.05) with GA3 application. It has been proposed that the effects of Rht8

are possibly due to reduced sensitivity to brassinosteroid.

Previous research has indicated that the main disadvantage of Rht12 is the long

vegetative phase resulting in late ear emergence.

Origin and History of Rht-B1b , Rht-D1b and Rht8

The GA-insensitive height reducing genes Rht-B1b and Rht-D1b originated in the 1930’s

in the dwarf variety Norin 10, a derivative of the Japanese variety Daruma (Allan, 1989).

Shortly after World War II, S. C. Salmon, a wheat breeder with the USDA, visited Japan

as an advisor to the occupation army. During his visit, he received several wheat samples,

and among them was the variety Norin 10, which he sent to the USDA Small Grains

Collection Facility. In 1948, Norin 10 was obtained by Orville Vogel, a USDAARS

wheat breeder in Pullman, WA, who then crossed Norin 10 with the high-yielding variety

Brevor 14 (Allan, 1989). The Norin 10 x Brevor 14 cross was then used by Norman

Borlaug and others as part of wheat improvement programs in the United States and at

the International Maize and Wheat Improvement Center (CIMMYT) (Ellis et al, 2002).

The Japanese variety Akakomugi was the source for most of the European cultivars

carrying the Rht8 dwarfing gene (Worland et al., 1998). Rht8 is widespread in southern

and central European wheats as well as several Russian cultivars (Worland et al., 1998)

Akakomugi was first used by the Italian breeder Strampelli in the 1920’s to introduce

genes not only for semi-dwarfism (Rht8) but also, unknowingly, for early maturity (Ppd-

D1) (Worland and Law, 1986, Korzun et al., 1998).

From Italy, Rht8 made its way to Argentina before World War II and and then to Europe

and the former Soviet Union after World War II (Borojevic and Borojevic, 2005). Unlike

Rht-B1b and Rht-D1b, the Rht8 dwarfing gene from Akakomugi is sensitive to exogenous

GA.

Wheat Norin 10

It is a semi-dwarf wheat cultivar with very large ears that was bred at an experimental

station in Iwate Prefecture, Japan. In 1935, it was registered as a numbered cultivar by

Ministry of Agriculture and Forestry .Norin 10 provided two very important genes, Rht1

and Rht2, that resulted in reduced-height wheats, thus allowing better nutrient uptake and

tillerage (when heavily fertilised with nitrogen, tall varieties grow too high, become top-

heavy, and lodge).

Genetics of Rht-B1b, Rht-D1b, and Rht8

Figure. : Comparison of the nucleotide sequences of the Rht-1 homeologs across the

conserved N-terminal coding region. SNPs between the Rht-1 homeologs are indicated

by lowercase letters and asterisks above the sequences .The Rht-B1b and Rht-D1b point

mutations are shown, and their positions are indicated by arrows above the sequences.

The predicted amino acid sequences of RHT-A1A, RHT-B1A, and RHT-D1A are

identical in this region.

Rht-B1 is located on chromosome 4B (Gale and Marshall, 1976), which was confirmed in

both hexaploid wheat (Rao, 1980) and durum wheat (Blanco et al., 1998).

Figure. :Rht-D1 is located on chromosome 4D (Gale et al., 1975).

Traditionally, selection for the Rht-B1b and Rht-D1b dwarfing genes was determined by

testing seedlings with gibberellic acid (GA). Plants with the mutant dwarfing gene show

no response to exogenous GA. Both Rht-B1b and Rht-D1b dwarfing genes are insensitive

to exogenous GA. With the introduction of marker-assisted selection, perfect markers for

Rht-B1b and Rht-D1b were developed in a doubled haploid population that was

segregating for the Rht-B1b and Rht-D1b alleles (Ellis et al., 2002).

Perfect markers detect the specific base-pair mutation responsible for the semi-dwarfing

phenotype.

The mutations involved in the Rht-B1b and Rht-D1b semi-dwarf phenotypes disrupt the

GA signaling pathway. The wild type proteins are thought to act as negative repressors of

GA signaling and GA acts by repressing their function (Hussain and Peng, 2003). Peng et

al. (1997) identified base substitutions in both Rht-B1b and Rht-D1b. In both cases, the

mutations affects the N-terminal region of their transcribed DELLA proteins via a

substitution which produces a stop codon shortly after the transcription start site (Peng et

al., 1997), resulting in their characteristic GA-insensitivity. DELLA proteins are a

subfamily of GRAS proteins, which are thought to act as transcriptional regulators (Pysh

et al., 1999). Rht-B1b and Rht-D1b produce stop codons in the DELLA domain, a 27

amino acid motif at the N-terminus, and in their truncated form, these proteins are

thought to act as constitutive repressors of GA-mediated growth (Peng et al., 1997).

Gale et al. (1982) found the location of a major Rht allele on chromosome 2D with a

backcross monosomic analysis using varieties Sava (derived from Italian wheats) and

Koga II. This allele accounted for nearly all of the height difference between the two

parent varieties. Another study, by Law et al. (1981), determined that the dwarfism of

Mara, an Italian variety derived from Akakomugi, was partially caused by chromosome

2D. The causal gene was later designated as Rht8. The microsatellite marker WMS 261 is

located 0.6cM distally from Rht8 can be used as a marker to identify lines carrying the

Rht8 dwarfing gene (Korzun et al., 1998).

Rht8 has been shown to be closely linked to the photoperiod-insensitivity gene Ppd-D1

(Korzun et al., 1998). Photoperiod-sensitive wheat varieties require long days for floral

induction, while photoperiod-insensitive varieties have the ability to flower

independently of photoperiod. Several studies have shown that varieties exhibiting

photoperiod-insensitivity also exhibit earlier heading and shorter stature than their

photoperiod-sensitive counterparts (Marshall et al., 1989; Blake et al., 2009). In addition

to effects on heading date and height, Dyck et al. (2004) found that photoperiod

insensitivity had a significant effect on yield. In the current study, lines were screened for

photoperiod-insensitivity and removed to reduce confounding effects.

2.12 Agronomic Traits of Rht-B1b, Rht-D1b, and Rht8

Figure : Most dwarfing mutations introduce premature stop codon

Height

The height reduction associated with Rht-B1b and Rht-D1b arises from GA insensitivity

that causes a decrease in cell elongation in juvenile leaf and stem tissue, which leads to an

overall reduction in plant height. Height reductions for cultivars carrying the Rht-B1b and

Rht-D1b dwarfing genes are similar to each other. Rht-B1b and Rht-D1b were found to

reduce plant height by 15% (Gale and Youseffian, 1985) and 24% (Allan, 1986). Another

study found a reduction of 14% and 17% for Rht-B1b and RhtD1b, respectively

(Flintham et al., 1997). Trethowan et al. (2001) reported an average height reduction of

36% in a population of Rht-B1b near-isogenic lines. Blake et al. (2009) suggested that

final plant height is influenced by not only genotype but also a variety of environmental

factors, such as heat, drought, and nutrient deficiencies. Rht8 has been shown to reduce

height by approximately 10% in studies from the UK, Germany, and former Yugoslavia

(Worland and Law, 1986; Worland et al., 1998). Reductions of 3.49% (Börner et al.,

1993), 7.3% (Rebetzke et al., 1999), and 12.5% (Rebetzke and Richards, 2000) have also

been reported.

Yield

Reports of the advantages and disadvantages of different Rht alleles and their standard

height counterparts have drawn varying conclusions. Increased yield potential for Rht-

B1b and Rh-D1b has been noted under high-input growing conditions (Knott, 1986;

Hedden, 2003; McNeal et al., 1972) Although Rht-B1b and Rht-D1b dwarfing genes

have the potential to increase yield of wheat grown in optimal conditions, these dwarfing

genes have been associated with reductions in yield in environments with low-inputs or

abiotic stresses (Laing and Fischer, 1977; Anderson and Smith, 1990; Richards,

1992) .The yield advantages of Rht-B1b and Rht-D1b are less obvious in spring wheat

than in winter wheat as well as in conditions of heat or drought stress (Flintham et al.,

1996). Heat and drought stress during ear initiation can reduce grain number through a

reduction in the number of competent florets and pollen viability and can reduce grain

weight as a result of shortened grain-fill period (Hoogendoorn and Gale, 1988).

Rht-B1b, Rht-D1b, and possibly Rht8, are associated with increased floret fertility which

may counteract the negative effects on yield observed with some Rht genes (Gale and

Youseffian, 1985). Yield increases in semi-dwarf wheat cultivars are due, in part, to

increased partitioning of assimilates into the developing grain rather than into the stem

for elongation (Flintham et al., 1997).

Several studies suggest that there is not a significant difference between Rht-B1b and

Rht-D1b in terms of yield improvement. In Montana and Saskatchewan trials, semidwarf

lines containing Rht-B1b or Rht-D1b generally yield more than standard height lines,

except in very low yielding environments, where the standard height lines exhibited a

yield advantage (Knott, 1986; McNeal et al., 1972). Yield increases of 24% (Flintham et

al., 1997) and 16% (Singh et al., 2001; Allan, 1986) have been reported for Rht-B1b and

Rht-D1b. Yield increases of 21% (Chapman et al., 2007) for Rht-B1b and 30% (Blake et

al., 2009) and 18% (Chapman et al., 2007) for Rht-D1b have been reported. For cultivars

carrying Rht8, yield increases of 12% (Gale etal., 1982), 9.7% (Rebetzke and Richards,

2000), and 3.8% (Börner et al., 1993) have been reported.

Coleoptile Length

Crop establishment is a major determinant of yield (Paulsen, 1987) and coleoptile length

is an important factor in seedling emergence and crop establishment. Reduced coleoptile

length is associated with reduced emergence and subsequent poor crop establishment

(Allan, 1980). In modern wheat cultivars, one of the most important determinants of

coleoptile length is the presence of the Rht semi-dwarfing genes.

Standard height (tall) wheats have long coleoptiles, due to normal cell elongation in the

presence of endogenous GA, and Rht-B1b and Rht-D1b semi-dwarf wheats, which are

GA-insensitive, have shortened coleoptiles (Keyes et al., 1989). Allan (1980) found that

Rht-B1b and Rht-D1b reduce coleoptile length in a similar proportion to their reduction

in plant height. Wheat cultivars carrying Rht-B1b and Rht-D1b dwarfing genes have a

limited coleoptile length of about 7.0 cm, while the coleoptiles of standard height wheats

can reach up to 13.0 cm (Whan, 1976). Another study, by Trethowan etal. (2001),

reported standard height average coleoptile length at 12.4 cm and Rht-B1b semidwarf

average coleoptile length at 7.8 cm.

In cultivars containing the GA-sensitive Rht8 dwarfing gene, there seems to be a

negligible effect on coleoptile length (Konzak, 1987). Additionally, Rebetzke et al.

(1999) reported Rht8 semi-dwarf coleoptiles as long as those of the standard height

parent.

Stem Solidness

The wheat stem sawfly, Cephus cinctus Norton, is a major insect pest of wheat and other

cereals across areas of western North America as well as Canada (Davis, 1955). Adults

deposit eggs in the stems and when the larva hatch, they feed on the parenchyma and

vascular tissue inside of the stem, moving down the stem until they reach near ground

level, where they cut around the inside of the stem. Then, they move down into the

remaining portion of the stem to pupate and overwinter (Hayat, 1993). Agricultural losses

caused by wheat stem sawfly are estimated at $25 million per year (Montana State

University, 1997). Currently, the main control method has been the use of solid stemmed

cultivars.

Stem solidness is caused by the development of undifferentiated parenchymous cells, or

pith, inside the stem. The thickness of the parenchymal cell walls has been reported to

have a direct relationship with larval mortality in wheats with solid stems (Roemhild,

1954). Thus far, no published studies have examined the effect of Rht dwarfing genes on

stem solidness. The current study examines the effect of Rht genotype on stem solidness.

Effects of Exogenous GA3 Application

Gibberellins (GAs) are a major class of plant hormones that regulate plant growth and

development, from seed germination and stem elongation to fruit-set and growth . It is

important for plants to produce and maintain optimal levels of bioactive GAs to ensure

normal growth and development. Mutants with impaired GA biosynthesis or response

show typical GA-deficient phenotypes, such as dark green leaves, dwarfism and late-

flowering, while elevated exogenous GA dose or increased signaling can cause excessive

plant growth and earlier flowering . Mutants deficient in GA biosynthesis can be rescued

by exogenously applied GAs but this is not possible if the mutation is in the GA signaling

pathway .

The deployment of genes influencing plant height through the GA pathway was a major

factor in the success of the Green Revolution, which created high-yielding cultivars of

rice and wheat with shorter and sturdier culms . In contrast to the recessive, semi-dwarf

sd-1 Green Revolution allele in rice, which is a loss-of-function mutation in one of the

major GA biosynthetic genes , the reduced height Rht-B1b (Rht1) and Rht-D1b (Rht2)

Green Revolution alleles in wheat are semi-dominant gain-of-function mutations causing

impaired GA signaling and thus conferring dwarfism through constitutive repression of

cell division and elongation .

The wheat Green Revolution genes are orthologues of the Arabidopsis GA-insensitive

(gai), the rice slender1 (slr1) or the rice gai , the barley slender1 (sln1) and the maize

dwarf-8 (d8) genes. However, in addition to reducing plant stature, Rht-D1b and Rht-

B1b also reduce seedling vigour and coleoptile length, and may reduce crop water-use

efficiency and performance in some unfavorable environments . So, opportunities exist

for replacing Rht-B1b and Rht-D1b in wheat with alternative dwarfing genes, such as the

GA-responsive dwarfing genes (Rht4, Rht5, Rht9, Rht12, Rht13, Rht14, Rht15, Rht16 or

Rht18). These genes have been reported to reduce plant height without compromising

early plant growth . Even though there are several GA-responsive dwarfing genes in

wheat, their molecular characteristics remain obscure and the mechanisms by which they

resulted in a reduction of plant height is not well understood. The metabolic pathways of

gibberellin biosynthesis, deactivation and signaling have become relatively clear and

many of the genes involved have been identified , which lays the foundation for analysis

of GA-responsive dwarfing genes in wheat.

Generally, dwarfing genes in wheat are classified into two categories, GA-responsive

(GAR) and GA-insensitive (GAI), reflecting the relative magnitude of their responses to

application of exogenous GAs . GA-responsive dwarfing genes show significantly

enhanced growth response to exogenous GAs (probably have mutations in GA

biosynthesis pathway) while GA-insensitive dwarfing genes show very little response to

exogenous GAs (probably have mutations in GA signaling pathway, such as Rht-D1b and

Rht-B1b) . This classification has usually been conducted at the seedling stage, for

example, based on the response of coleoptile length or the first seedling leaf elongation

rate to exogenous GAs . There is less information available on the response of the GAR

dwarfing genes to exogenous GAs at later growth stages. Rht12 significantly decreased

stem length (43% 48% for peduncle) and leaf length (25% 30% for flag leaf), while∼ ∼

the thickness of the internode walls and width of the leaves were increased. Additionally,

the Rht12 dwarf lines showed very dark green leaves compared to tall lines. Rht12

significantly decreased plant height, by around 40%, while seedling vigour, coleoptile

length and root traits at the seedling stage were not affected adversely. Rht12 lines had

significantly increased floret fertility and grain number and achieved a higher harvest

index (due to the lower plant biomass) than the tall genotypes. However, Rht12 extended

the duration of the spike development phase, especially the duration from sowing to

double ridge, and delayed anthesis date by around 5 days. . Another negative effect of

Rht12 on yield components was that grain size was reduced significantly. Similarly, other

studies have found that Rht12 had a substantial effect on reducing plant height without

altering early vigour and significantly increased spikelet fertility, harvest index, and

lodging resistance but these were usually accompanied by delayed ear emergence and

reduced grain weight. .Although Rht12 has been classified as a GA-responsive dwarfing

gene , a comprehensive understanding on the response of Rht12 to exogenous GAs is

lacking. Thus, the role of Rht12, if any, in GA biosynthesis or signaling is still unclear.

Moreover, it has been recently found that the effects of the dwarfing gene Rht8, which

had been considered as ‘GA-responsive’, was possibly not due to defective GA

metabolism or signaling because the wild type and Rht8 lines responded with a very

similar increase in final plant height (15% and 13%, respectively; P<0.05) with GA3

application. It has been proposed that the effects of Rht8 are possibly due to reduced

sensitivity to brassinosteroids .

DELLA Proteins

Figure8. : The amino acid sequence of DELLA protein

DELLA proteins are negative regulators of GA-induced growth. In the absence of GA,

DELLA proteins repress expression of GA response genes resulting in slow growth,

whereas with GA, there is induced phosphorylation of DELLA proteins via an

unidentified kinase. The SCFSLY1 complex interacts with the GRAS domain of DELLA

proteins and targets their polyubiquitination and degradation via the ubiquitin–26S

proteasome pathway (Dill et al., 2004). Small deletions of the DELLA protein that

interfere with degradation (Itoh et al., 2002, 2005; Liu et al., 2010), such as a gai mutant

of the Arabidopsis DELLA domain that lacks 17 amino acids near the N terminus, cause

dwarf stature (Fleck & Harberd, 2002) due to constant gene repression even in the

presence of GA. Similarly, a T-to-G substitution converts the E61 codon (GGA) to a

translational stop codon (TGA) in the Rht-D1b allele and the resultant N-terminally

truncated product confers short stature (Peng et al., 1999). Overexpression of DELLA

proteins is also a powerful way to reduce stature. Low-level gai expression caused

relatively mild height reduction, whereas high-level gai expression resulted in more

severe dwarfism in Arabidopsis (Fu et al., 2001).

Figure : The GA‐DELLA signalling mechanism. In the absence of GA, the DELLA

proteins are stabilized in the nucleus and repress DELLA‐mediated growth, presumably

via modulation of transcription of target genes. In the presence of GA, GA binds to the

soluble GID1 receptor. In the nucleus the GA‐GID1 complex associates with the DELLA

proteins, promoting a further interaction between DELLA and the SCFSLY1/GID2 complex.

The SCF complex catalyses the polyubiquitination of the DELLA protein, triggering

DELLA degradation by the 26S proteasome. Destruction of the DELLA protein

derepresses the DELLA‐mediated growth restraint and allows growth to occur.

DELLAs are a family of nuclear proteins that act as growth repressors throughout the life

cycle of higher plants. Derepression is mediated through the gibberellic acid (GA)‐

dependent degradation of DELLAs and the key components of the GA‐DELLA

signalling pathway (the GA receptor and the F‐box protein involved in DELLA

destruction) have recently been identified. It is becoming increasingly clear that DELLAs

promote a plant's survival by integrating its growth responses to a wide range of

endogenous and environmental signals.

Figure: DELLA proteins consist of functional GRAS and regulatory DELLAdomain at

the less conservative N-terminus of the protein .Conserved N-terminal regulatory region

consists of DELLA,LexLe and TVHYNP amino acid motif.In the C-terminus functional

domains are LR1 & LR2(Leucine rich regions);NLS(nuclear localization signal);SH2-

like(src homology 2 like domains) and VHIID,PFYRE,RVER,SAW are the amino acid

motifs.

DELLAs N-termini show high homology to each other between 34 and 84 % similarity

(Bolle, 2004). Mutations in these genes produce amino acid substitutions, deletions or

insertions in the N-terminal region of the translated DELLA protein (in wheat, Rht-B1b

and Rht-D1b alleles encode proteins that have deletion between DELLA and TVHYNP

motifs in LExLE region). These mutations affect binding to GA-receptor and GA

allowing accumulation of mutant DELLAs and repress growth. In the nucleus, DELLA of

wild type binds to GA-receptor (e.g. GID1 known for Arabidopsis), GA and SCF E3

ubiquitin ligase complex. Such a large complex is recognized by 26S proteasome and

destroyed. The disappearance of DELLA proteins stimulates GA responsive processes

such as seed germination, stem and root elongation, and fertility (Hirsh, Oldroyd, 2009).

In the absence of GA, or in the case of mutation in nucleotide sequence of DELLA

domen, the ubiquitination becomes impossible. Accumulation of the mutant DELLA

proteins cause continuous 171Della mutations in plants with special emphasis on wheat

growth inhibition and, accordingly, leads to agronomically advantageous dwarfed plant

height and improved straw strength by inhibition of stem cell elongation (Dalrymple,

1986; Flintham et al., 1997; Peng et al., 1999). As has been shown for barley embrio

(Gubler et al., 2002; http://plantcellbiology.masters.grkraj. org), DELLAs repress

transcription and processing of GAMYB gene (GA induced Amylase-beta), that is

transcriptional regulator of α-amylase gene regulatory elements called GARE (GA

response elements) and induce amylase gene expression.

Figure. : Model of regulation of amylase biosynthesis by DELLA proteins

1.DELLA SCF E3 ubiquitin ligase complex degradation starts after binding with GA

2.The promoter of GAYMB gene becomes active and GAYMB transcription factor is synthesised

3.GAYMB transcription factor activates the alpha amylase gene.

4. Alpha amylase and other hydrolytic enzyme are synthesised in rough endoplasmic reticulum and secreted by golgi body.

5.Secretory vessels go through the cell wall and alpha amylase starts the starch degradation in endosperm.

DELLAs inhibit growth by interfering with the activity of growth-promoting

transcription factors (Harberd et al., 2009). Mutants of wheat, barley, and rice, that are

affected in GA signaling, display an altered aleurone α-amylase response. For example,

dominant mutations at the homeoallelic wheat Rht-B1a and Rht-D1a loci confer

dwarfism and а reduced growth response to GA (Börner et al., 1996; Peng et al., 1999).

Severely dwarfing alleles, such as Rht-B1c, abolish the GA response of mutant aleurone

cells (Gale, Marshall, 1975; Ho et al., 1981; Börner et al., 1996). DELLAs are

conservative due to the essential role in plant cell. They help to establish GA homeostasis

by direct feedback regulation on the expression of GA biosynthetic and GA receptor

genes, and promote the expression of downstream negative components that are putative

transcription factors/regulators or ubiquitin E2/E3 enzymes (Zentella et al., 2007). In

addition, one of the putative DELLA targets, XERICO, promotes accumulation of

abscisic acid (ABA) that antagonizes GA effects. Therefore, DELLA may restrict GA-

promoted processes by modulating both GA and ABA pathways (Zentella et al., 2007).