Cytoplasmic male sterility in Maize

Cytoplasmic male sterility

Plant inability to produce functional pollen grains is known as male sterility. Male sterility can be determined by nuclear (genetic male sterility) or cytoplasmic (cytoplasmic male sterility - CMS) genes.

More than 150 plant species are successfully used in commercial production of hybrid seed.
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History
Rhoades (1933) was the first to describe CMS in Peruvian maize population Another source of male sterility, found in 1939 in Argentina, no longer exists today (Duvick, 1965).

Cytoplasmic male-sterile trait could be used to enable crosses without manual detasseling.
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CMS in maize
Three main CMS types have been described: CMS-T (Texas) (Rogers and Edwardson, 1952) CMS-C (Charrua) (Beckett, 1971) CMS-S (USDA) (Jones,1957) Distinguished by specific nuclear genes (Rf genes) that restore pollen fertility. PCR enable to distinguish the three major types of CMS in maize (Liu et al., 2002).
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Fig.1. Recombinant mtDNA regions of the CMS T, C and S (Liu et al., 2002).

 Three CMS types can be distinguished from one another and from the fertile (N) mitochondria based on molecular criteria.
Within the normal and each CMS type, subgroups have been identified according to various criteria, like mtDNA restriction enzyme and hybridization analyses (Newton, 1988).

The CMS-C type, for example, has been divided into three subgroups on the basis of differences in the banding pattern of mtDNA (Pring et al., 1980). SubgroupI - C Cytoplasm SubgroupII - RB,BB Cytoplasm SubgroupIII - ES Cytoplasm
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CMS in maize cont.. Borck and Walbot (1982)

CMS maize Normal maize

mtDNA

Restriction enzyme digestion pattern

Concluded : 80% of the normal mitochondrial genome is composed of unique sequences 50% of the mtDNA sequences are shared by normal, CMS-C, CMST, and CMS-S maize.
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CMS in maize cont..

In 1970, a pathogen, Bipolaris maydis caused leaf blight in maize produced with CMS-T genotypes Both male sterility and disease susceptibility traits appear to be closely linked and are associated with a mitochondrial gene, T-urf13

Interaction between fungal toxins and URF13, cause permeabilization of the inner mitochondrial membrane, accounts for the specific susceptibility to the fungal pathogens.

Restorer genes
Certain nuclear genes (Restorer of fertility Rf) can overrule male sterile effect of cytoplasm. Thus CMS plant carry appropriate Rf gene produce functional pollen even if there cytoplasm is male sterile. Rf also known as supressor of CMS phenotype
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Reversion to fertility
The reversion of CMS strain to male fertility is based on genetic change Reversion can be spontaneous or mutagen induced

Certain strains of CMS-S revert rather frequently to male fertility (than T & C). Both nuclear & cytoplasmic reversions are involved Nuclear genotype strongly influence frequency of CMS-S reversion e.g. Reversion of CMS-S to fertility in inbreed M825 occurs at high frequency than inbreed WB4
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CMS combined with Xenia in Plus-Hybrid System

CMS effect
No pollen production

Xenia effect
Change in phenotype of seed as a result of source of pollination: direct influence on colour, weight and composition of the maize kernel

Plus-Hybrid Effect

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Fig. 2. Diagram of the Plus-Hybrid effect. The third column refers to a system that is not practicable in the field due to uncontrolled pollination (Urs Weingartner, 2002) 12

Restoration systems
Sporophytic Restoration Systems: act prior to meiosis or in sporophytic tissue Gametophytic Restoration Systems: act after meiosis in microspores or pollen grains These differences lead to very different fertility patterns.

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Fig.3 Maize-CMS Restoration of fertility system: different classes of pollen grains are produced, but not all of them are viable (Rainer Messmer).
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Different types of CMS

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CMS-C
Fertility restoration is Sporophytic Rf4, Rf5, Rf6 are responsible for fertility restoration Isolated CMS-C mitochondria synthesize 17.5kD Polypeptide

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CMS-S
Fertility restoration is Gametophytic Rf3 (chr. 2) are responsible for fertility restoration Isolated CMS-S mitochondria synthesize several minor high M.wt polypeptide Plant Heterozygote for Rf3 (Rf3 rf3) produce half normal (Rf3) & half abortive pollen (rf3)
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CMS-S conti.....
CMS-S in maize characterized by presence of 2 autonomously replicating plasmid like element S1 & S2. They are not found in mitochondria genome of normal male fertile maize. The relationship between the presence of S1 and S2 plasmids and pollen sterility in CMS-S is not understood.

Cytoplasmic reversion of CMS-S from male sterile to male fertile phenotype associated with disappearance of S1 & S2.
Loss of plasmid is under nuclear influence & is not a characteristic of S cytoplasm. Unlike CMS-T and CMS-C spontaneous reversion to fertility occurs in CMS-S. This could be due to either nuclear mutations or gene rearrangements. (Newton 1988; Braun et al 1992; Williams et al 1992).
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CMS-T
Fertility restoration is sporophytic Rf1 (chr. 3) & Rf2(chr.9) are responsible for fertility restoration Isolated CMS-T mitochondria synthesize 13-kD polypeptide Although presence of the Rf1 is sufficient to reduce synthesis of the polypeptide, but the action of both Rf1 and Rf2 is required to restore full fertility This system is most reliable & stable of maize male sterile cytoplasm Plant Heterozygote for both restorer gene (Rf1 rf1 Rf2 rf2) produce normal pollen 1/4th of pollen from such plant carry both the alleles
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Differences in the three major types of sterile cytoplasm in maize

Trait
Anther exsertion
Microsporogenesis Tapetal

CMS-C
Nil to little
Disturbed Abnormal

CMS-T
Nil
Normal Normal

CMS-S
Sometimes exserted
Normal Normal

Restorer gene action

Fertility restoration Restorer gene number Susceptibility to B. maydis Spontaneous reversion

After microspore development

Sporophytic Rf4, Rf5, Rf6 No low

During early prophase

Sporophytic Rf1, Rf2 on chr. 3, 9 yes low

During pollen maturation

Gametophytic Rf3 on chr. 2 No High
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Cytological & Ultra structure studies

CMS-T
Cytological study on anther of CMS-T Mitochondria of tapetal cells undergoes change in ultra structure Followed by change in Mitochondria of microspore

CMS-C
Cytological study on anther of CMS-C Initial structural abnormalities in tapetal cells No Mitochondrial alterations observed

CMS-S
Cytological study on anther of CMS-S Development of CMS-S anther is identical to that of normal cytoplasm anther, till very late pollen development

Late breakdown of development of pollen is because of gametophyte mode of restoration

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How to determine CMS type?

Tester lines, containing nuclear restorer genes, were traditionally used to determine and classify the CMS types of maize. Due to the development of molecular methods in recent years, our understanding of the molecular basis of CMS has increased dramatically. A further goal of this work was to develop a PCR assay for the determination of cytoplasm types. PCR-based mitochondrial DNA markers were developed based on the chimeric DNA sequences in different male-sterile cytoplasms of maize.
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Mechanism of CMS
A number of cytological and molecular studies have already been carried out to investigate:
CMS mechanisms Understand the factors responsible for pollen abortion.

Several reports have highlighted the Importance of the tapetal layer of anthers in the development of viable pollen grains (Colhoun, 1981; Wen and Chase, 1999; Kapoor, 2002; Luo, 2006).
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Mechanism of CMS Conti.

CMS originated from mutations in the mtDNA (Because of aberrant intra/intermolecular recombination) Rearrangements modify existing genes/generate unique genes (chimeric genes)

These are transcriptionaly active

Expressed as novel mitochondrial proteins Cause failure in mitochondrial function in the tapetum and microspores leading to failure in the production of viable pollen
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Chimeric gene
The chimeric gene T-urf13 was detected in mtDNA of CMS-T (Dewey et al. 1986). ORF was found in a chimeric atp6 / atp9 region of cms-C (Dewey et al., 1991). A repeated mtDNA region R containing two chimeric ORFs-ORF355 & ORF77 found in cmsS (Zabala et al.,1997).
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(CMS-T) maize as model system for CMS

General information

Texas (T) cytoplasmic male sterility discovered in 1940s used extensively throughout the 1960s.
Highly stable under all environmental conditions. Characterized by failure of anther exertion and pollen abortion.

Plants bearing the T cytoplasm, are susceptible to southern corn leaf blight - (B.maydis)
Toxin produced by B.maydis = T-toxin. Susceptibility arises in T cytoplasm due to mitochondrial sensitivity to T-toxin. 26

T-urf13 gene in T cytoplasm maize

Mitochondrial gene T-urf13 is a unique chimeric sequence A 13 kDa polypeptide is observed in T-mitochondria, but not in N-mitochondria.

The gene encoding this polypeptide arose through mitochondrial recombination.

T-urf13 encodes a nonessential polypeptide. Amount of the polypeptide is reduced in presence of the Rf1 gene Dewey et al. (1987).
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How does URF13 cause cms?

Effect of URF13 protein Degeneration of the tapetum during microsporogenesis Disruption of pollen development leading to male cell abortion Why would mitochondrial disfunction specifically affect pollen development? Mitochondrial gene functions are essential to all cells electron transfer, ATP formation, and translation of mitochondrial mRNA. Interruption of any of these functions would be expected to be lethal.
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Conclusions
CMS employed for hybrid seed production in many plant species the molecular & functional bases for CMS Remain unclear. But the picture changed with advent of techniques like R/E cloning & sequencings technologies The combination of male sterility & xenia increase grain yield than produced by pure male fertile maize crops. Growing mixtures of male sterile & male fertile maize could be put into practice in near future
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