We reported previously the isolation of a novel cell death-suppressing gene from maize (Zea mays) encoded by the Lls1 (Lethal leaf spot-1) gene. Although the exact metabolic function of LLS1 remains elusive, here we provide insight into mechanisms that underlie the initiation and propagation of cell death associated with lls1 lesions. Our data indicate that lls1 lesions are triggered in response to a cell-damaging event caused by any biotic or abiotic agent or intrinsic metabolic imbalance-as long as the leaf tissue is developmentally competent to develop lls1 lesions. Continued expansion of these lesions, however, depends on the availability of light, with fluence rate being more important than spectral quality. Double-mutant analysis of lls1 with two maize mutants oil-yellow and iojap, both compromised photosynthetically and unable to accumulate normal levels of chlorophyll, indicated that it was the light harvested by the plant that energized lls1 lesion development. Chloroplasts appear to be the key mediators of lls1 cell death; their swelling and distortion occurs before any other changes normally associated with dying cells. In agreement with these results are indications that LLS1 is a chloroplast-localized protein whose transcript was detected only in green tissues. The propagative nature of light-dependent lls1 lesions predicts that cell death associated with these lesions is caused by a mobile agent such as reactive oxidative species. LLS1 may act to prevent reactive oxidative species formation or serve to remove a cell death mediator so as to maintain chloroplast integrity and cell survival.lls1 (lethal leaf spot-1) is a maize (Zea mays) mutation, characterized by the formation of necrotic spots that expand continuously to kill the entire leaf and eventually the whole plant. The developmentally programmed phenotype of lls1 manifests in a cell autonomous fashion as evidenced by the discrete border between mutant and revertant tissue in sectored plants (Gray et al., 1997) and is suggestive of the involvement of an endogenous program in lls1 cell death. Because this mutation is inherited in a strictly recessive fashion, it is likely that the wildtype Lls1 gene functions to positively maintain cell homeostasis (Ullstrup and Troyer, 1967; Johal et al., 1994). The Lls1 gene has been cloned. Although it appears to encode a novel protein specific to plants, it does have two motifs, a Rieske-type Fe-sulfur center and a mononuclear non-heme Fe-binding site, that are found in the aromatic ring-hydroxylating dioxygenases of bacteria. Because of the fact that the biochemical function of these enzymes is to degrade aromatic hydrocarbons, we hypothesized previously that LLS1 may also work by breaking down a phenolic mediator of cell death in plants (Gray et al., 1997). This proposal remains contentious, however, because the nature of the substrate, if any, for LLS1 remains unknown and we have now found these motifs in a small family of plant enzymes, two of which are known to function in chlorophyll b and Gly betai...

SummaryAll above-ground plant organs are derived from shoot apical meristems (SAMs). Global analyses of gene expression were conducted on maize (Zea mays L.) SAMs to identify genes preferentially expressed in the SAM. The SAMs were collected from 14-day-old B73 seedlings via laser capture microdissection (LCM). The RNA samples extracted from LCM-collected SAMs and from seedlings were hybridized to microarrays spotted with 37 660 maize cDNAs. Approximately 30% (10 816) of these cDNAs were prepared as part of this study from manually dissected B73 maize apices. Over 5000 expressed sequence tags (ESTs) (about 13% of the total) were differentially expressed (P < 0.0001) between SAMs and seedlings. Of these, 2783 and 2248 ESTs were up-and down-regulated in the SAM, respectively. The expression in the SAM of several of the differentially expressed ESTs was validated via quantitative RT-PCR and/or in situ hybridization. The up-regulated ESTs included many regulatory genes including transcription factors, chromatin remodeling factors and components of the gene-silencing machinery, as well as about 900 genes with unknown functions. Surprisingly, transcripts that hybridized to 62 retrotransposon-related cDNAs were also substantially up-regulated in the SAM. Complementary DNAs derived from the LCM-collected SAMs were sequenced to identify additional genes that are expressed in the SAM. This generated around 550 000 ESTs (454-SAM ESTs) from two genotypes. Consistent with the microarray results, approximately 14% of the 454-SAM ESTs from B73 were retrotransposon-related. Possible roles of genes that are preferentially expressed in the SAM are discussed.

Microarrays enable comparative analyses of gene expression on a genomic scale, however these experiments frequently identify an abundance of differentially expressed genes such that it may be difficult to identify discrete functional networks that are hidden within large microarray datasets. Microarray analyses in which mutant organisms are compared to nonmutant siblings can be especially problematic when the gene of interest is expressed in relatively few cells. Here, we describe the use of laser microdissection microarray to perform transcriptional profiling of the maize shoot apical meristem (SAM), a ~100-μm pillar of organogenic cells that is required for leaf initiation. Microarray analyses compared differential gene expression within the SAM and incipient leaf primordium of nonmutant and narrow sheath mutant plants, which harbored mutations in the duplicate genes narrow sheath1 (ns1) and narrow sheath2 (ns2). Expressed in eight to ten cells within the SAM, ns1 and ns2 encode paralogous WUSCHEL1-like homeobox (WOX) transcription factors required for recruitment of leaf initials that give rise to a large lateral domain within maize leaves. The data illustrate the utility of laser microdissection-microarray analyses to identify a relatively small number of genes that are differentially expressed within the SAM. Moreover, these analyses reveal potentially conserved WOX gene functions and implicate specific hormonal and signaling pathways during early events in maize leaf development.

The maize (Zea mays) shoot apical meristem (SAM) arises early in embryogenesis and functions during stem cell maintenance and organogenesis to generate all the aboveground organs of the plant. Despite its integral role in maize shoot development, little is known about the molecular mechanisms of SAM initiation. Laser microdissection of apical domains from developing maize embryos and seedlings was combined with RNA sequencing for transcriptomic analyses of SAM ontogeny. Molecular markers of key events during maize embryogenesis are described, and comprehensive transcriptional data from six stages in maize shoot development are generated. Transcriptomic profiling before and after SAM initiation indicates that organogenesis precedes stem cell maintenance in maize; analyses of the first three lateral organs elaborated from maize embryos provides insight into their homology and to the identity of the single maize cotyledon. Compared with the newly initiated SAM, the mature SAM is enriched for transcripts that function in transcriptional regulation, hormonal signaling, and transport. Comparisons of shoot meristems initiating juvenile leaves, adult leaves, and husk leaves illustrate differences in phase-specific (juvenile versus adult) and meristem-specific (SAM versus lateral meristem) transcript accumulation during maize shoot development. This study provides insight into the molecular genetics of SAM initiation and function in maize.

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