A variety of mutants have arisen in Mutator active lines which are being used at the University of Missouri to select photosynthesis (hcf) mutants. One such very interesting mutant has appeared in the second generation after outcross of Robertson Mutator stocks. This mutant segregates as a normal single recessive gene. Its primary phenotype is a loss (non-development) of leaf blade tissue. The absence of leaf blade due to this mutant gene can vary from a complete absence of leaf blade leaving only the midrib to a 10% loss of tissue from the extreme margins of the leaves. The gene is now being named leaf bladeless (lbl) for the most obvious phenotype.
It appears that normal development is expressed in the first 3 to 5 leaves rather than the lbl phenotype. Leaf tissue first appears not to developed normally from this juvenile stage to maturity in a temperature sensitive manner. The mutant gene (lbl) is expressed at temperature above approximately 24 C, however, near-normal leaf blade development occurs at temperatures below about 21 C. In our experiments plants show near-normal leaf development when grown in the winter greenhouse, but lbl is maximally expressed in summer field culture in Missouri. Summer plants produce only leaf midrib tissue but no leaf blade. This tissue becomes trapped inside the older tissue and often produces a tangled mass which is unable to elongate unencumbered. This results in a plant no more than 20 to 30cm high. Plants grown at less than 21 C in the winter greenhouse often produce an ear shoot and a small tassel. However we have not yet been able to collect viable seed from lbl/lbl plants.
The expression of lbl in plants in the 21 to 24 C range shows a range of expression in development of blade tissue. Five levels have been designated for non-development of leaf blade tissue in the leaves of these plants. All of these different stages can be observed in one individual plant.
Stage 1. In the most extreme case of lbl expression there is nothing more than the midrib of the leaf, which results in a cylindrical structure from 3 to 10mm in diameter (Figure 1B). This cylindrical structure has well-developed vascular tissue in the central location of the structure with a layer of parenchyma surrounding it. In the parenchyma there are 10 to 20 leaf vascular bundles complete with vascular tissue and a green bundle sheath cell layer. The midrib cylinder is surrounded by apparently normal epidermal tissue with normal appearing green guard cells. There is no chlorophyll in any of the other tissue of this structure other than the bundle sheath. The green vascular bundle strands have well-developed lamellar chloroplasts on a clear white parenchyma background. The vascular bundles appear to be separated by about 1mm. This provides an interesting, well developed tissue which contains only green bundle sheath cells, and only bundle sheath chloroplasts with no contamination with mesophyll chloroplasts. This could represent an important experimental tissue for C4 photosynthesis.
Figure 1. Expression of the leaf blade-less mutant gene (lbl) in growth of maize leaves at 24 C.
Stage 2. In the second level of expression we see the midrib with small amount of leaf blade tissue (less than 1.0cm wide) attached (not shown). This generally takes two forms which are classified as (2a), the blade tissue developed for the full length of the leaf or (2b), the blade tissue developed only on the basal 25% of the midrib tissue. This small amount of blade tissue has normal mesophyll cells and mesophyll chloroplasts.
In both stage 1 and 2 we always see a demarcation of the sheath location with a ligule. In stage 1 the ligule may be the only non-midrib tissue which develops.
Stage 3. In this stage the midrib is accompanied by a near normal half-leaf blade (Fig. 1B). That is, we observe leaf blade tissue on one side of the midrib but none on the other side. In this case the blade tissue is normally attached to the midrib throughout the length of the organ. Stages 1, 2, and to some extent, stage 3 occur at temperatures of 24 C or higher.
Stage 4. In stage four of expression of lbl, the midrib is separated from the blade tissue throughout the distal 1/3 to 1/2 of the length of the leaf. There is a clear gap of tissue which did not develop between the vascular tissue and the blade allowing the separation of the leaf into 2 or 3 different linear structures. The development of the two lateral halves of the leaf is usually not correlated. This is shown by the leaves in Fig. 1A, C, and D. In Fig. 1D, there are three distinct structures, the midrib, the left blade half and the right blade half. The gap between the foliar structures appears to be a lack of tissue development rather than abnormal tissue development. At the point the detached leaf blade halves meet the midrib there appears to be a small knot of tissue.
Stage 5. In stage five the leaf appears near normal except that as much as 10% of the lateral margins is missing or abnormally developed (not shown). Often this appears as undulating, abnormally expanding leaf blade tissue at the margins.
It appears clear that the normal function of the lbl gene and the site for lbl/lbl is at the shoot meristem in the early stages of leaf primordia development or development of the intercalary meristem leading to the development of the leaf tissue. The mutant gene expression only becomes apparent after the seedling leaves have developed. It could be suggested that the parent plant provides a diffusible factor to override lbl/lbl and allow normal leaf development for up to the first five leaves. When the embryo germinates this factor is no longer available and incomplete leaf development is observed provided the seedling is exposed to an environmental temperature of 24 C or higher.
The development of leaf tissues and the final shape of the leaf result from the system of cell division and elongation from the meristem. When leaf initiation occurs from the apical shoot meristem, the leaf develops from a meristematic cell mass which gives rise to the basal intercalary meristem (Sharman, B. C., Ann. Bot. 6:245-284, 1942). Clonal analysis indicated that leaf cells come from at least two layers (LI and LII) in the shoot meristem (Poethig, S., in Contemporary Problems in Plant Anatomy, White and Dickison, eds, 1984). LI (protoderm) produces the leaf epidermis complete with guard cells. This layer must be fully active in even the most extreme stages of lbl expression because a normal epidermis is always present. The LII layer gives rise to the remainder of the leaf including the vascular tissue, the bundle sheath and the mesophyll. Dengler et al. (Amer. J. Bot. 72:284-302, 1985) have shown that the bundle sheath cells and the mesophyll develop from different cell lineages, the bundle sheath and the vascular bundle originating from the procambium and the mesophyll from the ground meristem. Further, Langdale et al. (Genes and Develop. 2:106-115, 1988) have presented data supporting an independent development of mesophyll and bundle sheath cells.
These observations mesh well with the developmental leaf patterns observed with lbl plants. In stage 1 of development the protoderm and the procambium yield a cylindrical structure completely devoid of mesophyll tissue. In later stages the ground meristem becomes active providing from a very small amount (stage 2) to a near normal amount (stage 5) of mesophyll tissue. In stage 3 the ground meristem is active on only one side of the procambium (Figure 1B) and in stage 4, there are three points of activity, one at the procambium and two in the ground meristem (Figure 1 D) which later join to form a complete leaf for the basal portion.
Moreover, lbl regulates the activity of the ground meristem in a temperature sensitive manner. These explanations are a suggested starting point for future analysis of this mutant.
A further interesting feature of the mutant stock is that it was induced
in a Mutator line. Therefore, it is possible that it was Mutator induced
and potentially the gene could be cloned using the Mutator probe.
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