Genetic Dissection of Root Formation in Maize (Zea mays) Reveals Root‐type Specific Developmental Programmes

Abstract

• Background Maize (Zea mays) forms a complex root system comprising embryonic and post‐embryonic roots. The embryonically formed root system is made up of the primary root and a variable number of seminal roots. Later in development the post‐embryonic shoot‐borne root system becomes dominant and is responsible together with its lateral roots for the major portion of water and nutrient uptake. Although the anatomical structure of the different root‐types is very similar they are initiated from different tissues during embryonic and post‐embryonic development. Recently, a number of mutants specifically affected in maize root development have been identified. These mutants indicate that various root‐type specific developmental programmes are involved in the establishment of the maize root stock.

• Scope This review summarizes these genetic data in the context of the maize root morphology and anatomy and gives an outlook on possible perspectives of the molecular analysis of maize root formation.

INTRODUCTION

Root systems of terrestrial plants serve many important tasks among which anchorage of the plant and uptake of water plus nutrients are the most important ones (Aiken and Smucker, 1996).

Cereals including maize (Zea mays) account for about 70 % of the human calorie intake worldwide (Chandler and Brendel, 2002). Despite their agronomic importance a systematic genetic analysis of root formation in cereals has only recently been initiated (Feix et al., 2002; Hochholdinger et al., 2004), although the genetic potential of the maize root system has already been recognized more than 70 years ago: ‘There is every reason to believe that the root systems also should exhibit large numbers of heritable characters’ (Jenkins, 1930).

This review will summarize the current status of the genetic analysis of maize root development, and starts with a concise introduction of the morphological and anatomical features of the maize root system.

MORPHOLOGY OF THE MAIZE ROOT STOCK

Maize belongs to the family Poaceae (formerly known as Gramineae). The organization of seedlings from this family is so distinct from other families of monocotyledonous plants that a whole set of specific terms describing their seedling structures (e.g. scutellar node, coleoptilar node, mesocotyl or coleorhiza; Fig. 1) have been introduced (Tillich, 1977).

Fig. 1. The maize root system at different stages of development, illustrating the consecutive emergence of the four major root types: (A) 3 dag (days after germination); (B) 5 dag; (C) 10 dag; (D) adult above‐ground root system about 6 weeks after germination. The different root types are labelled in capital letters, other terms describing specific features of the maize seedling are labelled in lower case. PR, primary root; SR, seminal root; CR, crown root; BR, brace root; co, coleorhiza; c, coleoptilar node; m, mesocotyl; s, scutellar node.

The root system of maize can be divided into an embryonic root system (Abbe and Stein, 1954) consisting of a single primary root and a variable number of seminal roots, and a post‐embryonic root system which is made up by shoot‐borne roots. Shoot‐borne roots formed at consecutive underground nodes are called crown roots, while the respective roots formed at consecutive above‐ground nodes of the shoot are called brace roots (Fig. 1). Lateral roots which emerge from all major root‐types also belong to the post‐embryonic root system.

Embryonic root formation

In contrast to the primary roots of most angiosperms which are formed exogenously (i.e. from the outermost cell layer of the embryo), the primary root of maize (Fig. 1A) is formed endogenously (Yamashita, 1991; Yamashita and Ueno, 1992) deep inside the maize embryo and becomes visible as a distinct region of the embryo about 10–15 d after pollination. Endogenously formed primary roots are found in no other family outside the Poaceae (Tillich, 1977). The term endogenous implies that the root tip, including the root cap, of maize has to penetrate and break other tissues before it becomes visible. The tissue that is damaged during primary root emergence forms the so‐called coleorhiza (Fig. 1A) which encloses the proximal end of the newly developed primary root (Tillich, 1977).

Seminal roots which emerge from the scutellar node (Fig. 1B) are also formed endogenously and become visible in the embryo between 22–40 d after pollination (Sass, 1977; Erdelska and Vidovencova, 1993; Feldman, 1994). Seminal roots do not form a coleorhiza since the scutellar node tissue is already differentiated when seminal roots emerge and can easily be penetrated. The number of seminal roots per seedling is variable between 0 and 13 and strongly dependent on the genetic background of the seedling (Kiesselbach, 1949; Sass, 1977; Feldman, 1994).

Primary and seminal roots can persist and remain functional during the whole life cycle of the maize plant (e.g. Kiesselbach, 1949; Kausch, 1967; Kozinka, 1977; McCully and Canny, 1985). Some authors have observed that the primary and seminal roots die after the formation of the shoot‐borne root system (e.g. Lawson and Hanway, 1977; Feldman, 1994). That the primary root and its lateral roots alone are sufficient to form a fertile mature plant was demonstrated by the monogenic recessive mutant rtcs (Hetz et al., 1996), which forms only a primary root and its lateral roots but no seminal or shoot‐borne roots. This mutant will be discussed later in the section on genetic analysis of maize root formation.

Early post‐embryonic root formation

Traditional descriptions of maize morphology distinguish between embryonic and post‐embryonic root development. As a result of the recently identified mutants that will be described later, we suggest that post‐embryonic root development is subdivided into an early and a late phase which are regulated by different genes.

During the first 2 weeks of development the embryonic primary and seminal roots make up the major portion of the seedling rootstock. Later, the post‐embryonic shoot‐borne roots become dominant and form the major backbone of the maize root system. Early post‐embryonic root development is characterized by two root‐types: the lateral roots which emerge from the primary and seminal roots about 6–7 d after the formation of these main roots and the shoot‐borne crown roots that are formed at the coleoptilar node about 10–14 d after germination (Fig. 1C). Lateral roots have a strong influence on root architecture (Lynch, 1995) and are responsible for the major part of water and nutrient uptake of the maize plant (McCully and Canny, 1988; Wang et al., 1991; Varney and Canny, 1993; Wang et al., 1994) due to their branching capacity which leads to lateral roots of second, third and higher order. These roots, which are also sometimes referred to as branch roots, differ from the main roots of maize in that they are mostly very short (Varney et al., 1991), are more responsive to drying by transpiration (Wang et al., 1991), lose their meristem and become determinate very soon (Varney and McCully, 1991) and have an open late metaxylem for most of their length (Wang et al., 1994). Their open late metaxylem is responsible for the dominant role of these roots in water uptake (Wang et al., 1995). Lateral roots are initiated in the differentiation zone of pre‐formed roots after dedifferentiation of already differentiated pericycle cells (Esau, 1965). Whether the pericycle cells that give rise to lateral roots are already differentiated in maize is still under debate because the time interval between meristem exit of these pericycle cells and lateral root initiation was found to be relatively short (8.3 h, Dubrovsky and Ivanov, 1984), leaving little if any time for dedifferentiation. In maize, endodermal cells are also involved in lateral root formation (Bell and McCully, 1970), giving rise to the newly formed epidermis and columella, while the remaining root‐types are formed by the pericycle (Fahn, 1990). An exact prediction of which pericycle cells will divide is difficult (Charlton, 1991).

Shoot‐borne root formation mainly belongs to late post‐embryonic root development. However, the mutant lrt1 (which will be described later) indicates that crown root formation at the first node is genetically related to lateral root initiation in the embryonic root system.

Late post‐embryonic root formation

Crown roots are endogenous, like all shoot‐borne roots, and their primordia are formed opposite to collateral vascular bundles (Martin and Harris, 1976). Late post‐embryonic root development is characterized by the development of shoot‐borne crown and brace roots. The maize rootstock develops about 70 shoot‐borne roots during its life cycle, which are organized on average in six whorls of underground crown roots and two to three whorls of above‐ground brace roots (Hoppe et al., 1986). The transition between early and late post‐embryonic root development starts about 3–4 weeks after germination with the formation of whorls of crown roots at the second and consecutive nodes. The mean diameter and number of shoot‐borne roots per whorl increases at higher nodes (Hoppe et al., 1986). Since the first four underground internodes are very short (Hoppe et al., 1986), these nodes are located closely together forming a dense rootstock. Crown roots at lower nodes grow first horizontally before they follow the gravitropic vector, while roots from higher nodes grow directly downwards (Feldman, 1994). The crown roots form the major part of the adult rootstock and are the basis for lodging resistance of the plants. They are also responsible for the majority of the water uptake by the plant via their branch roots (McCully and Canny, 1988).

The aboveground‐formed shoot‐borne brace roots are also formed endogenously, like all roots of maize (Fig. 1D). Not all brace roots penetrate into the soil. Brace roots form lateral roots only after they have penetrated the soil and provide additional lodging resistance and water and nutrient uptake via their branch roots (Feldman, 1994).

In addition to the roots that are determined by the endogenous developmental programme, maize can also develop roots that are formed under unusual conditions such as wounding, hormone application or other exogenous stimuli at uncharacteristic places of the maize plant, for instance at the mesocotyl. In maize, these are commonly referred to as adventitious roots. Traditionally, all roots that are not derived from another root are described as adventitious. In maize, this classical definition would therefore also include all shoot‐borne roots plus the primary root. To avoid the confusion of specifying different root types with the same term we would suggest calling exogenously induced roots adventitious, and shoot‐borne roots, depending on their under‐ or aboveground position on the shoot, crown and brace roots, respectively.

FUNCTIONAL ANATOMY OF MAIZE ROOTS

Radial organization

Mature primary and seminal roots as well as shoot‐borne roots show a polyarch organization, which means that they exhibit a central cylinder (protostele) with many xylem arms. The mature primary root shows six to ten metaxylem elements, while the largest shoot‐borne roots can contain up to 48 metaxylem elements (Tillich, 1992) with an increasing number of metaxylem arms in shoot‐borne roots at successively higher nodes (Hoppe et al., 1986). The number of protoxylem elements is variable. Usually, two to three protoxylem strands, which alternate with the primary phloem, are arranged per metaxylem element (Feldman, 1994). The pericycle forms the outermost layer of the central cylinder (Fig. 2A). The ground tissue consists of one layer of endodermal tissue with the casparian strip, which represents a barrier of variable resistance to the radial flow of water and nutrients (Hose et al., 2001), and several layers (8–15) of parenchymatous cortex tissue (Fig. 2A). The outermost cell layer is formed by the epidermis (sometimes designated as rhizodermis), which consists of root‐hair‐forming trichoblasts and non‐root‐hair‐forming atrichoblasts. Arrangement of tricho‐ and atrichoblasts is irregular so that predictions as to which cells will form root hairs are difficult. In older roots the short‐living epidermis is replaced by a lignified, suberinized exodermis, which develops from the outermost cells of the cortex and forms an additional casparian band (Feldman, 1994). In aboveground‐formed brace roots the epidermis persists and forms a protective cuticula. Maize roots, like those of most monocotyledonous plants, do not show secondary growth of the root.

Fig. 2. Anatomical structure of the maize primary root, displaying the relative positions of the different cell types: (A) cross‐section; (B) median longitudinal section. The images are light microscopic photographs that have been coloured. After Hochholdinger et al. (2004). Longitudinal zones of development are indicated at the right margin of (B): D, division; E, elongation; M, maturation.

Transverse organization

Traditionally, the longitudinal structure of the maize root is described in terms of various partially overlapping specialized zones of development including the root cap, the root apical meristem, the distal elongation zone, the elongation zone and the maturation zone (Ishikawa and Evans, 1995) (Fig. 2B). The root cap covers the root tip and secrets mucilage which facilitates the movement of the growing roots in the soil. Proximal to the root cap is the subterminal root apex, which consists of the quiescent centre (QC), a mitotically inactive region of 800–1200 cells (Jiang et al., 2003) and is surrounded by the proximal and distal meristems (Fig. 2B). Next to the proximal meristem is the distal elongation zone in which the newly generated cells start to elongate. The distal elongation zone is a transition zone between the meristematic zone and the elongation zone in which cells are difficult to classify according to their mitotic activity or allometric coefficient of expansion (Ishikawa and Evans, 1995). This zone plays an important role in the response of maize roots to a variety of exogenous environmental signals (summarized in Ishikawa and Evans, 1995). The distal elongation zone is bordered by the elongation zone, where cells do not divide any more but elongate maximally. Further proximal to the elongation zone is the maturation zone where differentiation of the cells, indicated by root hairs (Ishikawa and Evans, 1995), is observed.

This traditional two‐dimensional view of root organization is appropriate to explain the basic anatomical structure of the maize root. However, it does not reflect the actual three‐dimensional situation, which is more realistically described in terms of cylinders, sectors, packets and cell files as suggested by Rost and Bryant (1996). These aggregates can be correlated with unique signals and gene expression patterns and can therefore be regarded as key developmental units which integrate the complex networks of interacting developmental signals.

GENETIC DISSECTION OF ROOT FORMATION IN MAIZE

Factors that complicate the genetic analysis of maize root formation

Traditional forward genetic approaches identify new monogenic mutants from large F₂ populations that have previously been mutagenized by the visual assessment of aberrant phenotypes of certain organs or developmental stages in segregating families. In maize, mutagenesis is usually performed via transposon tagging (Walbot, 2000) or pollen mutagenesis with chemicals such as EMS (ethyl methane sulfonate) (Neuffer, 1994). This approach has been successfully applied to a number of aboveground traits of maize (e.g. Coe et al., 1988; Sheridan and Clark, 1988) but, for several reasons, is not as straightforward when adapted to the analysis of root development.

First, roots grow in soil and are not directly accessible for phenotypic analyses. Isolation of complete rootstocks from soil is difficult because it is almost impossible to do so without damaging them. A screening procedure that allows the dissection of large numbers of intact maize root systems in a reasonable time is necessary. Germination of maize kernels in paper rolls (e.g. Hetz et al., 1996) is a non‐invasive method that allows the study of undamaged root systems under standardized conditions. Until about day 14 after germination maize seedlings can live completely on the nutrients stored in their endosperm and can therefore be grown in distilled water under very stringently controlled temperature, humidity and light conditions in a growth chamber.

Secondly, the root system of mature maize plants has a considerable size and complexity (Kiesselbach, 1949). Therefore, a suitable developmental stage that provides maximum structural information and a minimum of complexity has to be chosen. Young maize seedlings about 10–14 d after germination already display three of the four major root‐types, namely the primary, seminal and crown‐roots (Fig. 1C). In addition, lateral roots which are common to all root‐types can already be studied at this developmental stage. Finally, these young seedlings are still very small so that about 1000 seedlings can be grown per square meter in a growth chamber.

Thirdly, root architecture is very susceptible to changes in environmental conditions. Root–soil interactions, which are mediated by a structure called the rhizosheath, strongly influence root architecture (McCully, 1999). The rhizosheath, which contains tightly bound soil particles associated with roots via their root hairs, integrates the information of the biotic and abiotic environment and makes this information available to the roots. However, roots are not only influenced by environmental stimuli but can also dictate their biotic environment via their border cells. Border cells are cells that are separated from the periphery of the root cap into the root environment (Hawes et al., 1998). These cells disperse into mucilage, which can attract or repel certain microorganisms within the immediate surroundings of the root. Local availability of nutrients can also have dramatic influences on root architecture. Thus changing phosphorus or nitrate availability, for example, can dramatically affect root branching, as observed in barley (Drew and Saker, 1978, 1975). The plethora of external cues that can influence root architecture is another reason to analyse seedlings at an early developmental stage under standardized conditions in distilled water.

Quantitative root traits are often inherited in a polygenic way

Another difficulty when working with agronomically relevant traits is that many quantitative traits are not inherited in a monogenic manner but are controlled by multiple genes (Stuber, 1995). Examples of polygenically controlled root traits in maize are seminal root number, dry weight, shoot : root ratio or root pulling resistance (O’Toole and Bland, 1987). Quantitative trait loci (QTL) analyses have recently been initiated to map some of these polygenically controlled traits (e.g. Tuberosa et al., 2002a, b).

Several root mutants of maize are pleiotropic

In addition to the observation that several quantitative root characters are controlled by more than one gene, pleiotropic inheritance has also been reported quite often with regard to root characters in maize. Pleiotropy means that many monogenic mutants that show an aberrant root phenotype also display an entire series of visible morphological changes in other parts of the plant. This phenomenon can be observed during all phases of maize development. Sheridan and Clark have identified a large number of maize embryogenesis mutants. Several of these emb mutants display defects in the primary root but also in other parts of the embryo (e.g. Clark and Sheridan, 1991; Sheridan and Clark, 1993; Clark, 1996). Among the defective seedling mutant series isolated by Gavazzi and co‐workers (e.g. Gavazzi et al., 1993; Dolfini et al., 1999) several mutants (des11, des21, des24) display defects in their seedling roots but are also affected in the green part of the seedling. Finally, the early phase change mutant (epc) of maize, which displays a reduced duration of the juvenile phase, also shows reduced shoot‐borne root formation (Vega et al., 2002).

Monogenic root mutations of maize often show a root‐type specific phenotype

In recent years several maize mutants with an aberrant phenotype that is confined to root development have been identified (Feix et al., 2002). Interestingly, many of these mutants show a root‐type specific phenotype in that they do not affect all major root types (Hetz et al. 1996; Hochholdinger and Feix, 1998a; Hochholdinger et al., 2001; Woll and Hochholdinger, 2004).

These mutants can be classified spatially or temporally. The spatial classification distinguishes between mutants of shoot‐borne root, lateral root or root hair formation, according to the root‐types affected. A temporal classification is based on the developmental phase that is affected in these mutants.

Mutants of shoot‐borne root formation.

Thus far, two mutants of maize affected in shoot‐borne root formation have been isolated. The mutant rt1 was the first mutant of root formation that was isolated (Jenkins, 1930) and shows a reduced number of shoot‐borne roots. The rt1 mutant is missing all shoot‐borne roots at the higher nodes (nodes 7 and 8) while there is only a slight difference in the number of crown roots at the first two nodes (Jenkins, 1930). This might indicate that the mutant rt1 mainly affects the above‐ground shoot‐borne root system while only slightly affecting the early underground shoot‐borne roots. The mutation rt1 is inherited as a monogenic recessive trait and maps on chromosome 3 (Emerson et al., 1935).

In contrast, the mutant rtcs (Hetz et al., 1996) (Fig. 3A and B) is completely devoid of all shoot‐borne roots plus the embryonic seminal roots. The only main root‐type that remains in rtcs is the primary root. In rtcs the primary root and its lateral roots are sufficient to generate a mature plant that can be propagated, if lodging is avoided, by tying the plant to a stick during development. The mutation inhibits the initiation of the affected root‐types, as indicated by histological sections of embryos and nodal tissue, and cannot be rescued by exogenous application of auxin (Hetz et al., 1996). The notion that no cell division occurs in the affected nodal tissue is also supported by the observation that none of the four maize cyclins, cyc1a, cyc1b, cyc2 and cyc3 (Renaudin et al., 1994) is expressed in the coleoptilar node (Hochholdinger and Feix, 1998b). However, the expression of cdc2a (Colasanti et al., 1991), which is a marker of competence for cell division (Colasanti et al., 1993), shows the same expression in the coleoptilar node of rtcs seedlings as in wild‐type seedlings (Hochholdinger et al., 1998b). Moreover, a cross between gaspe flint, an inbred line that shows excessive tillering from lower nodes as a dominant trait, and rtcs results in an F₂ generation that shows the rtcs phenotype and also tillering at the lower nodes (Fig. 3C) (Hochholdinger and Feix, 1998c). These results indicate that the mutation in the rtcs gene does not inhibit cell division in general in shoot nodes, although root formation from these nodes is suppressed. The rtcs mutation maps to the short arm of chromosome 1 (Hetz et al., 1996; Krebs et al., 1999). The map positions of rt1 and rtcs on different chromosomes indicate that the affected genes are not allelic.

Fig. 3. Recently identified monogenic recessive mutants of maize. (A) Close‐up of young 10‐d‐old wild‐type (left) and rtcs (right) seedlings. While the wild‐type forms seminal and crown roots, the rtcs seedling displays only a primary root with lateral roots, which will remain the only roots formed in this mutant. (B) Adult field‐grown rtcs shoot. No brace roots are formed at any node. (The wild‐type situation is displayed in Fig. 1D.) (C) The rtcs mutant introgressed into the in‐bred line gaspe flint. The primary root remains the only root type in this 6‐week‐old plant; however, lateral shoots develop from lower nodes. (D) Wild‐type (left) and slr1 (right) seedlings. The mutant slr1 forms lateral roots which are significantly shorter than in the wild type. (E) Wild‐type (left) and slr2 (right) seedlings. As in slr1, the lateral roots of the mutant are significantly shorter than in the wild type. (F) Wild‐type (left) and lrt1 (right) seedlings. In lrt1 lateral root formation is blocked before initiation. (G) Wild‐type (left) and rum1 (right) seedlings. The mutant rum1 is affected in lateral and seminal root initiation. (D–G) All seedlings are 14 d old. A detailed description of the mutants is given in the text. Table 1 summarizes the affected root types of these mutants.

Mutants of lateral root formation.

Recently, four mutants of maize affected in lateral root formation have been isolated. Two of the lateral root mutants, lrt1 (Hochholdinger and Feix, 1998a; Fig. 3F) and rum1 (Woll and Hochholdinger, 2004; Fig. 3G), are affected before lateral root initiation. The mutants slr1 (Hochholdinger et al., 2001; Fig. 3D) and slr2 (Hochholdinger et al., 2001; Fig. 3E) are both impaired in lateral root elongation which results in a reduced length of the affected lateral roots compared with the wild‐type situation. A common feature of all four mutants is that lateral root formation is only affected in the embryonic primary and seminal roots but not in the shoot‐borne root system. In lrt1 lateral roots cannot be induced by auxin (Hochholdinger and Feix, 1998a). However, inoculation with the arbuscular mycorrhizal fungus Glomus mosseae or growth in a high phosphate environment induces lateral roots in this mutant (Paszkowski and Boller, 2002). In rum1, in addition to the missing lateral root formation on the primary root, no primordia for the embryonic seminal roots can be detected during embryogenesis (Woll and Hochholdinger, 2004). For slr1 and slr2 normal root initiation was observed. The primordia of these mutants cannot be distinguished from their wild‐type counterparts. However, after normal lateral root primordia and meristems are established these lateral roots show reduced longitudinal elongation to about one‐quarter of the corresponding wild‐type cortical cells (Hochholdinger et al., 2001).

Mutants of root hair formation.

Root hairs are unicellular structures of the epidermis that play an important role in water and nutrient uptake (Schiefelbein, 2003). Thus far, three mutants of maize with abnormal root hair morphology have been described (Wen and Schnable, 1994). The mutant rth1 initiates normal‐looking root hair primordia that fail to elongate. The mutant rth2 also initiates normal root hair primordia that elongate to about 20–25 % of the normal wild‐type length. The mutant rth3 also fails to elongate the root hairs like rth1 but already seems to be disturbed during the establishment of the root hair primordium. While the mutants rth2 and rth3 grow normally, the mutant rth1 displays nutrient deficiencies. This might indicate that, under some environmental conditions, root hairs might be dispensable. Normal growth of rth2 and rth3 is also surprising since the rhizosheath structure which facilitates communication at the root–soil interface, i.e. the nutrient and water uptake from the soil, is reduced or missing in these mutants.

Monogenic root mutants of maize define different phases of post‐embryonic root development.

Instead of classifying monogenic root mutants of maize according to the root‐type that is affected in these mutants, they can also be classified according to the developmental phase in which the mutation becomes visible (Table 1). When this type of classification is applied, development of individual roots and development of the whole root system has to be distinguished.

Table 1.

Summary of the root types that are affected in the monogenic root‐specific mutants of maize identified thus far

1

Red indicates root types that are completely missing in the affected mutants. Grey indicates root types that are formed but are affected by the mutation. For the rth mutants all affected roots show impaired root hair formation but are otherwise normal (T. J. Wen and P. S. Schnable, pers. comm.). P, S, C and B indicate on which root type lateral roots are affected: P, primary root; S, seminal roots; C, crown roots; B, brace roots.

The developmental phases on the level of individual roots are grouped into root initiation, elongation and differentiation (Ishikawa and Evans, 1995). According to this scheme, the mutants rtcs (Hetz et al., 1996), lrt1 (Hochholdinger et al., 1998a) and rum1 (Woll and Hochholdinger, 2004) are affected in root initiation. The mutants slr1 and slr2 (Hochholdinger et al., 2001) are affected in root elongation, and the root hair mutants rth1, rth2 and rth3 (Wen and Schnable, 1994) are affected in root differentiation.

If mutations are classified on the level of the whole root system, development can be defined in terms of embryonic, early post‐embryonic and late post‐embryonic root development (Table 1). The mutants rtcs and rum1 are both affected in embryogenesis in that they fail to initiate seminal roots. Both mutants also show a post‐embryonic phenotype. While rum1 is affected in early post‐embryonic root development, which is represented by lateral root formation on the primary root, the mutant rtcs shows a complementary phenotype in that the lateral roots on the primary root are formed normally but all later post‐embryonic roots, including all shoot‐borne roots, are missing.

The mutants slr1 and slr2 are affected in lateral root formation of early post‐embryonic lateral roots formed at the primary and seminal roots. The mutant lrt1 defines early post‐embryonic root development even more precisely. In this mutant, no embryonic defect is detectable. Again, initiation of the lateral roots at the embryonic roots, which represent the early post‐embryonic root development, is affected. However, this mutant also shows problems with the initiation of shoot‐borne roots at the coleoptilar node. Although this phenotype is sometimes leaky, it might indicate that shoot‐borne root formation at the first node could be controlled by some factors that are also controlling lateral root formation at the embryonic roots. Since in this mutant only lateral roots formed at embryonic, but not at post‐embryonic, roots are affected, it might indicate that shoot‐borne root formation at the coleoptilar node and higher nodes may be regulated by alternative pathways. The mutant rt1 is, according to this scheme, mainly affected in late post‐embryonic root development. While crown root formation at the coleoptilar node, which belongs to the early post‐embryonic phase, is only slightly reduced, late post‐embryonic shoot‐borne roots are more severely reduced. Interestingly, lateral root formation at the shoot‐borne roots is regulated independently from that of the embryonic roots in all mutants identified thus far. Currently no mutant is available which shows any lateral root defects at the shoot‐borne roots. The root hair mutants rth1, 2 and 3 affect all root types and therefore all developmental phases except the brace roots, which do not form root hairs but instead develop a protective cuticula.

Interaction of monogenic root loci in maize: analysis of double mutants

A double mutant analysis of the mutants rtcs, lrt1, slr1 and slr2 (Table 2 and Fig. 4) reveals that the rtcs locus that controls the formation of the shoot‐borne root system acts independently of the other three loci (Table 2). Double mutants of rtcs with lrt1, slr1 and slr2 formed a primary root as the only major root‐type (Fig. 4A–C). The lateral roots of this single root were either missing (in combination with lrt1, Fig. 4C) or reduced in their elongation (in combination with slr1 and slr2, Fig. 4A and B). F₂ progenies of the cross lrt1 with slr1 and slr2 displayed, besides wild‐type seedlings, either lrt1 and slr1 or lrt1 and slr2 phenotypes. Segregation ratios (not shown) indicated that lrt1 might be epistatic to slr1 and slr2 (Table 2). The combination of the loci slr1 and slr2 resulted in a completely different phenotype from the single mutants (Table 2 and Fig. 4D). Lateral root elongation is further reduced and primary and seminal roots are shorter and show a disturbed longitudinal pattern of the cortical cells (Hochholdinger et al., 2001). This different phenotype becomes even more distinct 25 d after germination (Fig. 4E). This novel phenotype of the double mutant might indicate that these loci cooperate in the establishment of lateral roots in the embryonically formed root system, and also play a role in pattern formation of the embryonic roots after germination. In summary, the four monogenic recessive mutants of maize that have thus far been subjected to a double‐mutant analysis result in additive, epistatic and novel phenotypes giving an idea of the complex genetic networks that are involved in the establishment of the maize root system.

Table 2.

Summary of the root double‐mutants analysed thus far in maize, classified according to their phenotype

Interaction
of	with	Phenotype
rtcs	lrt1, slr1, slr2	Additive
lrt1	slr1, slr2	Epistatic
slr1	slr2	Novel

Additive and novel phenotypes are displayed in Fig. 4.

Fig. 4. Double‐mutant phenotypes of rtcs, lrt1, slr1 and slr2 that are additive or novel (Table 2). In A–D a wild‐type seedling (left), two single mutants and the double‐mutant (right) are depicted. The seedlings in each part are from one segregating F₂ family 14 d after germination. Details of the double‐mutant phenotypes are described in the text. From left to right: (A) wild type, slr1, rtcs, double mutant slr1/rtcs; (B) wild type, rtcs, slr2 double mutant slr2/rtcs; (C) wild‐ type, rtcs, lrt1, double mutant lrt1/rtcs; (D) wild type, slr2, slr1, double mutant slr1/slr2; (E) wild type (left) and double mutant (right) 25 d after germination. Note the well‐developed primary, seminal and lateral roots of the wild type compared with the short primary and seminal roots of the double‐mutants which do not display lateral roots.

PERSPECTIVES

Recently, a number of specific maize mutants affected in various aspects of root formation became available. These mutants will be a valuable source and starting point for a detailed study of the genetic networks involved in the establishment of the maize rootstock. However, one has to be aware that the genetic analysis of root formation in maize, i.e. the identification of monogenic root mutants, has not been exhaustive yet and that additional root mutants are to be expected in further screens. In addition, one also has to consider that the variability of root formation only allows for the identification of phenotypes that are drastically different from the wild‐type situation, i.e. that usually lack of one or several root types. Polygenic influences on root formation and pleiotropic phenotypes also limit the number of root‐specific mutants. Most of the mutants known so far have been isolated from seedling screens under standardized conditions. Alterations of the screening procedure, e.g. addition of particular exogenous stimuli such as hormones or nutrients, might reveal additional genetically induced root phenotypes. It will also be a challenge to identify mutants at later stages of development. Thus far, most mutants have been identified at the seedling stage. Therefore, for example, only lateral root mutants which were affected in lateral root formation at the young stage (i.e. primary and seminal roots) have been isolated (Hochholdinger and Feix, 1998a; Hochholdinger et al., 2001; Woll and Hochholdinger, 2004). It would be interesting to identify additional mutants that are defective in lateral root formation in the shoot‐borne roots, or in shoot‐borne root formation in general. A possible way to find such mutations during late root development could be the screening of altered above‐ground traits that might be the consequence of root deficiencies. Reduced lodging resistance or smaller plants in field experiments might be indicative of such defects. However, caution has to be exercised using this approach since transposon‐tagged populations show a high phenotypic variation in their above‐ground appearance. It might therefore be more promising to perform such screens in uniform inbred populations that have been mutagenized with chemical agents such as EMS which induce point mutations. A saturated mutagenesis of the maize root system might nevertheless be difficult to achieve because, due to the variability of root formation, only drastic phenotypes can be identified.

Thus far, only a few genes specifically expressed in maize roots have been cloned (e.g. Goddemeier et al., 1998; Matsuyama et al., 1999; Ponce et al., 2000). The specific root mutants that are now available are a starting point for a detailed molecular analysis of maize root formation. Resources such as high resolution genetic and physical maps, BAC collections anchored to these maps, and the initiation of the maize genome sequencing project (Bennetzen et al., 2001; Chandler and Brendel, 2002) will allow for the identification of genes affected in these mutants. For some of the mutants cloning of the mutated genes is already under way. Transcriptome‐ (Bruce et al., 2001) and proteome‐wide (Chang et al., 2000) approaches that compare gene expression and protein accumulation in maize wild‐type and mutant roots will also allow identification of downstream components involved in maize root formation, and will help to add new genes to the map that are involved in maize root formation. Various new technologies including laser capture microdissection now allow the study of gene expression of specific cell types of maize (e.g. Nakazono et al., 2003; Schnable et al., 2004). The generation of mutants via the reverse genetic knock‐out of root‐specific genes will further enhance the functional analysis of root formation in maize. In summary, the combination of classical genetic approaches together with newly emerging molecular techniques will allow for an extensive analysis of the genes that are involved in the development of the maize rootstock.

ACKNOWLEDGEMENTS

We thank Drs W. F. Sheridan and J. K. Clark (University of North Dakota) for advice on maize embryogenesis, Drs T. J. Wen and P. S. Schnable (Iowa State University) for discussions on root hairs, N. Höcker (University of Tübingen) for comments on the manuscript and Dr G. Feix (University of Freiburg) for continued support. Root research in F.H.’s laboratory is supported by the DFG (German Scientific Foundation) award HO2249/4‐1, the SFB 446 and the framework programme ‘heterosis in plants’ (award HO2249/6‐1).

Received: 17 November 2003; Returned for revision: 14 December 2003; Accepted: 16 December 2003; Published electronically: 23 February 2004