The auricles are two wedge-shaped structures that develop on either side of the midrib, expanding in the proximal-distal plane as they approach the margins. Auricles are derived from both epidermal and mesophyll tissue and their structural function is likely to allow the blade to bend outwards to maximise light capture. Together, the mature ligule and auricle represent the position of the blade/sheath boundary , but their position is defined by this boundary, not the other way round. The BSB is visible before ligule and auricle development and the anastomosis of intermediate veins, which also characterises the BSB, is visible in leaf mutants liguleless1and lg2 that lack the ligule. As such, it is clear that the BSB is a fundamental component of the leaf upon which the ligule and auricle are elaborations. The lg1 mutant is a classical mutant that lacks both ligule and auricle and possesses a diffuse BSB. The lg2 mutant is similar to lg1, but upper leaves have partial recovery of ligule and auricle at the margins, while lg1 does not display this partial recovery but has a slightly better defined boundary. lg1 leaves are slightly narrower than normal siblings. Tassel branch number is reduced in both lg1 and lg2 and the transition to the reproductive phase is delayed in lg2 mutants, such that they produce more leaves than normal siblings.The accumulation of LG2 mRNA precedes that of LG1 – it is observed in the meristem,grow racks with lights while LG1 mRNA is only observed at the BSB. On the other hand, LG2 mRNA is observed in the lg1 background and LG1 mRNA is observed in the lg2 background at the leaf margins, so there does not seem to be a direct “switching on” of LG1 by LG2 nor vice versa.

LG1 encodes a nuclear localised protein that contains a domain with significant similarity to SQUAMOSA PROMOTER-BINDING1 and SBP2 proteins from Antirrhinum majus. In contrast to LG2, LG1 functions cell-autonomously. Wavy auricle in Blade1is a dominant mutation that results in ectopic expression of the WAB1 protein in the leaf. WAB1 functions in the tassel to turn on LG1, which in turn promotes branch initiation. When WAB1 is ectopically expressed in the leaf, the resulting activation of LG1 causes the distinctive over proliferation of the auricle at the ligule boundary as well asectopic auricle and sheath in the leaf blade. At the blade sheath boundary the blade width of Wab1 is similar to normal siblings, but distal to this, the blade quickly narrows such that Wab1 has a very narrow blade further up the leaf. This narrowing is due to a deletion of the lateral domain of blade, as shown by analysis of the triple mutant ns1;ns2;Wab1, where both the marginal domain and lateral domains are lost. A reduction in the number of lateral veins in the Wab1 blade support the hypothesis that narrowing in the Wab1 blade is due to deletion of domains rather than loss of cell expansion59. The disruption of the BSB in lg1 and lg2 made them obvious candidates for crossing to fun. As Wab1 displays hypertrophy of the auricle, and fun displays complete loss of the auricle, we made a double mutant population to ask if FUN is required for ectopic auricle growth in the Wab1 background.Families segregating for Wab1, lg2, lg1 and fun were made according to the genealogy shown in Figure 3-1. Leaf width measurements were taken at blade mid point and/or 5cm above the blade/sheath boundary. Sheath lengths were measured from the base of the leaf to the MLK at the midrib and/or the auricle at the margin closest to the base of the leaf.

Plants were identified by their distinctive phenotypes with double mutant phenotypes from previous generations informing identification of plants as more mutations were introduced into the family. In families AV814-7, genotypes of double and triple mutants were also deduced from the presence of single mutant plants in the family.In families AV182, AV545 and AV814-7, Wab1;fun plants were observed . Wab1 plants had slightly shorter blades than fun or normal siblings, while all three had similar sheath lengths. When Wab1 and fun were combined, blade lengths were reduced and sheath length was increased , suggesting a change in the position of the blade/sheath boundary . The increase in sheath length did not make up for the loss of blade, resulting in a leaf that was shorter overall in the double mutant. The plants had extremely narrow leaves at the midpoint – narrower than either Wab1 or fun alone . Wab1;fun double mutant plants have a disrupted BSB with both auricle and ligule displaying deviations from normal plants. While both Wab1 and fun have normal ligules, in the double mutant, the ligule is patchy, disrupted and displaced over a large region . Although disruption of the BSB in the Wab1;fun double mutant is extensive, the ligule at the intersection of the BSB and the midrib is consistently present and this point is also marked on the abaxial side of the leaf as a lighter coloured area which forms a raised bump along the midrib . I have termed this point the “midrib ligular knot” . In normal plants, the MLK is slightly proximal to the stem, compared to the marginal auricle, while in Wab1 plants the MLK and marginal auricle are about equidistant. In fun, the MLK is displaced distally compared to normal siblings while in the Wab1;fun double, the MLK is much more proximal to the base of the leaf than the marginal auricle .

Wab1;fun double mutants form points of ectopic ligule in the blade, reminiscent of the ectopic auricle in Wab1 single mutants, interestingly, there is very little, if any, ligule associated with the ectopic auricle in Wab1, while the double shows clear ectopic ligule . The Wab1;fun double mutant often retains some auricle at the margins of the leaf, and this is associated with “wings” of marginal tissue that extend a relatively short distance along the blade . The tassel of Wab1;fun plants resembles a fun tassel . Combining the Wab1 mutant with lg1 resulted in total loss of both the marginal auricle and MLK, though the boundary was still detectable by the commencement of the midrib . When Wab1 was combined with lg2, the MLK was lost but the marginal auricle was increased as in a Wab1 plant . In family AV545, the blade width at the midpoint was slightly reduced in lg2 plants but there was no evidence of an additive effect of narrowing in the Wab1;lg2 double mutant plants in family AV364 . A Wilcoxon t-test detected a reduction in width at the base of the leaf in Wab1;lg2 plants,rolling benches for growing though reference to the scatter plot reveals that the data are so scattered that the Wilcoxon t-test is misleading – rather I would argue that there is no difference in the width at the boundary between any of these mutant backgrounds. Tassel branch number is reduced to lg2 levels in the Wab1;lg2 double mutant . The Wab1;lg2;fun triple mutant showed loss of the extended marginal auricle seen in Wab1;lg2 as well as loss of the MLK seen in Wab1;fun .Both Wab1 and fun lead to narrowing of the leaf blade. Since narrowing in the leaf blade of Wab1 is due to a deletion of the lateral domain of the leaf, and narrowing in fun is associated with narrow cells , an additive interaction in the double mutant is not surprising. The lack of narrowing in lg2 and the epistatic narrowing observed in the lg2;fun double mutants implies that lateral expansion of the blade is associated with elaboration of the auricle but not definition of the MLK or the ligule itself. The partial recovery of blade width close to the BSB in the Wab1 mutant where there is extensive auricle supports this hypothesis.Although Wab1 leaf blades are slightly shorter than normal siblings, the extreme shortening of Wab1;fun double mutant blades was surprising since fun blades are of normal length . Furthermore, though Wab1 displays almost no displacement of the MLK and marginal auricle location as compared to normal siblings, and fun displays a slight distal displacement of the MLK, the Wab1;fun double mutant MLK is extremely proximal to the stem as compared to the marginal auricle .

This extensive synergistic interaction is mirrored in the Wab1;lg1 double but not the Wab1;lg2 double mutant. Since Wab1 is known to activate LG1 but not LG2 , these interactions would place FUN downstream of LG1. The additive interaction between lg2 and fun resulting in a loss of ligule at the midrib as well as loss of auricle at the margins shows that these genes function in different zones and pathways to elaborate the BSB. It seems likely that LG2 is responsible for elaborating the boundary at the midrib, while FUN elaborates the auricle at the margins. The different zones of action are underlined by the double mutants with Wab1 – loss of LG2 does not affect the elaboration of marginal auricle in the Wab1;lg2 double mutant , while the Wab1;fun double retains the MLK but loses the marginal auricle. In the Wab1;lg2;fun triple mutant both the marginal auricle is reduced, and the MLK is lost, as would be expected if LG2 and FUN work in different domains. It is possible that LG2 and FUN have a common upstream element and Two characterised mutants that are deffective in the synthesis of brassinosteroids in maize are nana1 and nana2. The maize mutant nana1 was isolated from the F2 of a plant with active Mutator transposons. PCR revealed a 497-bp insertion in a gene homologous to DE-ETIOLATED2in A. thaliana. DET2 is a steroid 5α-reductase known to catalyse a step in the BR synthesis pathway in A. thaliana. The knockout det2 phenotype can be rescued by addition of active brassinolide and is characterised in A. thaliana by extreme dwarfism as well as increased male sterility, dark green leaves, and repression of hypocotyl etiolation in the dark. In the maize na1 plant, levels of the substrate of the DET2 enzyme, -24- methylcholest-4-en-3-one, accumulated to 475% that of normal sibs, while downstream intermediates were reduced. While rescue by addition of brassinolide was not reported, addition of the BR inhibitor propiconazole to wild-type maize plants caused them to phenocopy the nana1 phenotypes of greatly shortened stature and feminised tassels. Since NA1 transcript was shown by in situ to accumulate in developing anthers, the role of BR in development of masculine flowers is supported. Five years after the publication of the causative mutation of the nana1 plant, the classical mutant nana2 was cloned31. Like na1, na2 is of greatly reduced height and has a feminised tassel. Since the na2 phenotype was so similar to na1, it was not surprising that a mutation was found in the maize orthologue of the A. thaliana DWF1 gene31. AtDWF1 is a Δ4-sterol reductase and dwf1 mutants accumulate its substrate 24-methylenecholesterol and have low levels of campestrol indicating a block in the BR pathway. The dwf1 phenotype, like det2, can be rescued by application of brassinolide in A. thaliana. The maize na2 plant was also shown to have low campestrol and later intermediates implying a similar block in the BR biosynthesis pathway. The overall na2 phenotype is similar to na1: short stature, feminised tassel and suppressed tillering. NA2 transcript is expressed in many tissue types including seedlings, mature and growing leaves, and immature ears and tassels with the highest expression detected in developing leaf collars, which is the position of ligule and auricle. Older tissues tend to have lower expression of NA2, supporting a role for BR in expansion and growth. Crosses with GA deficient mutants and application of GA to developing na2 plants has shown an interaction between these two hormone pathways in regulating growth and defining sex in Zea mays. GA and BR are thought to have independent roles in defining the absolute height of a plant, since the na2;d5 double mutant was shorter than either single mutant31. Masculine inflorescence development requires GA and BR to work together – the feminised tassel of na2 is abolished in the double mutant, implying that GA is needed for BR deficiency dependent feminisation. This hierarchy is mirrored in the ear: the anther ear phenotype of d5 is not abolished in the na2;d5 double mutant. Together these results show that BR is not essential for the masculinisation of inflorescences, while GA is essential for emasculation.