Judy Yamaguchi

A knotted1-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants.

The homeobox gene knotted1 (kn1) was first isolated by transposon tagging a dominant leaf mutant in maize. Related maize genes, isolated by virtue of sequence conservation within the homeobox, fall into two classes based on sequence similarity and expression patterns. Here, we report the characterization of two genes, KNAT1 and KNAT2 (for knotted-like from Arabidopsis thaliana) that were cloned from Arabidopsis using the kn1 homeobox as a heterologous probe. The homeodomains of KNAT1 and KNAT2 are very similar to the homeodomains of proteins encoded by class 1 maize genes, ranging from 78 to 95% amino acid identity. Overall, the deduced KNAT1 and KNAT2 proteins share amino acid identities of 53 and 40%, respectively, with the KN1 protein. Intron positions are also fairly well conserved among KNAT1, KNAT2, and kn1. Based on in situ hybridization analysis, the expression pattern of KNAT1 during vegetative development is similar to that of class 1 maize genes. In the shoot apex, KNAT1 transcript is localized primarily to the shoot apical meristem; down-regulation of expression occurs as leaf primordia are initiated. In contrast to the expression of class 1 maize genes in floral and inflorescence meristems, the expression of KNAT1 in the shoot meristem decreases during the floral transition and is restricted to the cortex of the inflorescence stem. Transgenic Arabidopsis plants carrying the KNAT1 cDNA and the kn1 cDNA fused to the cauliflower mosaic virus 35S promoter were generated. Misexpression of KNAT1 and kn1 resulted in highly abnormal leaf morphology that included severely lobed leaves. The expression pattern of KNAT1 in the shoot meristem combined with the results of transgenic overexpression experiments supports the hypothesis that class 1 kn1-like genes play a role in morphogenesis.

Isolation and expression of an anther-specific gene from tomato

SummaryWe have isolated and sequenced an anther-specific cDNA clone and a corresponding genomic clone from tomato. The gene (LAT52) encodes an 800-nucleotide-long transcript that is detectable in pollen, anthers and at 20-to 50-fold lower levels in petals. LAT52 mRNA is not detectable in pistils, sepals or non-reproductive tissues. Steady-state levels of LAT52 mRNA are detectable in immature anthers containing pollen at the tetrad stage and increase progressively throughout microsporogenesis until anthesis (pollen shed). The LAT52 gene contains 5′ and 3′ untranslated regions of 110 and approximately 150 nucleotides, respectively, and a single intron with a highly repetitive sequence. A TATA box motif is located 28 nucleotides upstream of the transcription start site. The gene encodes a putative protein of 18 kDa that is cysteine rich and has an N-terminal hydrophobic region with characteristics similar to eucaryotic secretory signal sequences. LAT52 is a single or low copy gene in tomato and shares homology with sequences in tobacco.

Sequence analysis and expression patterns divide the maize knotted1-like homeobox genes into two classes.

The homeobox of the knotted1 (kn1) gene was used to isolate 12 related sequences in maize. The homeodomains encoded by the kn1-like genes are very similar, ranging from 55 to 89% amino acid identity. Differences outside the precisely conserved third helix allowed us to group the genes into two classes. The homeodomains of the seven class 1 genes share 73 to 89% identical residues with kn1. The four class 2 genes share 55 to 58% identical residues with kn1, although the conservation within the class is greater than 87%. Expression patterns were analyzed by RNA gel blot analysis. Class 1 genes were highly expressed in meristem-enriched tissues, such as the vegetative meristem and ear primordia. Expression was not detectable in leaves. The class 2 genes were expressed in all tissues, although one was abundantly expressed in roots. The genes were mapped using recombinant inbred populations. We determined that clusters of two to three linked genes are present on chromosomes 1 and 8; otherwise, the genes are distributed throughout the genome. Four pairs of genes, similar in both sequence and expression patterns, mapped within duplicated regions of the genome.

Leaf Senescence Is Delayed in Tobacco Plants Expressing the Maize Homeobox Gene knotted1 under the Control of a Senescence-Activated Promoter

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.

Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia

A set of cDNAs that are expressed in tomato anthers were isolated [24]. We further characterized two of these cDNAs (LAT56 and LAT59) and their corresponding genomic clones. LAT56 and LAT59 show low levels of steady-state mRNA in immature anthers and maximal levels in mature anthers and pollen. The LAT56 and LAT59 genes are single-copy in the tomato genome, and are linked on chromosome 3, approximately 5 cM apart. Although these cDNAs did not cross-hybridize, their deduced protein sequences (P56 and P59) have 54% amino acid identity. The LAT56 and LAT59 genes each have two introns, but they are located in non-homologous positions. P56 and P59 show significant protein sequence similarity to pectate lyases of plant pathogenic bacteria. The similarity of P56 and P59 to the bacterial pectate lyases is equivalent to the homology described for different pectate lyase sequences of the genus Erwinia. We suggest that the pollen expression of LAT56 and LAT59 might relate to a requirement for pectin degradation during pollen tube growth.

Gametophytic and sporophytic expression of anther-specific genes in developing tomato anthers.

The tissue localization of transcripts corresponding to five anther-specific cDNA clones isolated from tomato was determined. Transcripts specified by three of the cDNA clones were first detectable in anthers containing mitotic-stage gametophytes and were localized to the gametophyte. Transcripts specified by the two other cDNA clones were not detectable until anthers had reached a later developmental stage; these transcripts were also localized to the (now bicellular) gametophytes. Transcript levels for all of the cDNAs increased during gametogenesis and reached maximal levels in mature pollen grains. These mRNAs persisted in in vitro-grown pollen tubes, concentrating toward the tips of the growing tubes. At flower maturity, transcripts specified by each of the cDNAs were also detected in the epidermal and endothecial cell layers of the anther wall. The spatial distribution of transcripts in the anther wall was confined to that region of the anther that surrounds the locule. Transcripts were not detected in the sterile tip of the anther or in the filament. mRNA levels for these cDNA clones were markedly reduced in the anthers of several independent male-sterile mutants of tomato. Our results provide evidence that these anther-specific cDNAs represent genes expressed in both the gametophytic and sporophytic phases of the plant life cycle. The patterns of mRNA accumulation observed support the hypothesis that the proteins encoded by these genes function during pollen development and pollen tube growth.

Gnarley1 Is a Dominant Mutation in the knox4 Homeobox Gene Affecting Cell Shape and Identity

Maize leaves have a stereotypical pattern of cell types organized into discrete domains. These domains are altered by mutations in knotted1 (kn1) and knox (for kn1-like homeobox) genes. Gnarley (Gn1) is a dominant maize mutant that exhibits many of the phenotypic characteristics of the kn1 family of mutants. Gn1 is unique because it changes parameters of cell growth in the basal-most region of the leaf, the sheath, resulting in dramatically altered sheath morphology. The strongly expressive allele Gn1-R also gives rise to a floral phenotype in which ectopic carpels form. Introgression studies showed that the severity of the Gn1-conferred phenotype is strongly influenced by genetic background. Gn1 maps to knox4, and knox4 is ectopically expressed in plants with the Gn1-conferred phenotype. Immunolocalization experiments showed that the KNOX protein accumulates at the base of Gn1 leaves in a pattern that is spatially and temporally correlated with appearance of the mutant phenotype. We further demonstrate that Gn1 is knox4 by correlating loss of the mutant phenotype with insertion of a Mutator transposon into knox4.

DELETION ANALYSIS OF POLLEN-EXPRESSED PROMOTERS'

SummaryWe have used microprojectile bombardment of tobacco pollen to study the DNA sequences involved in the expression of pollen-expressed genes. Promoter-reporter gene fusions constructed with the promoters of three different pollen-expressed genes from tomato (LAT52, LAT56, and LAT59) and either theβ-glucuronidase or luciferase reporter genes were assayed by bombarding hydrated tobacco pollen with the gene constructs precipitated onto tungsten microprojectiles. Reporter gene expression can be assayed within 30 min, with the maximal level of expression between 6 and 12 h after bombardment. By constructing and assaying promoter deletion derivatives, we have been able to delimit regions of the promoters that are necessary for high level expression in pollen. We also demonstrate that results with this transient expression system parallel the expression levels seen in pollen from stably transformed transgenic plants. The microprojectile bombardment assay can be used to rapidly test constructs for pollen expression beforeAgrobacterium-mediated plant transformation. Furthermore, it may be possible to adapt the microprojectile bombardment technique to achieve stable transformation of pollen.

Genetic and Molecular analyses of pattern formation genes in maize

One of the most salient features of pattern formation in plants is the extended period over which it occurs. Unlike animal development, in which the many of the large scale patterns are established during embryogenesis, many key aspects of a plant’s architecture are determined much later. In most seed plants, virtually the entire shoot system forms from a small group of cells contained in the shoot apical meristem (Steeves and Sussex, 1989). Although this meristem forms during embryogenesis, the initials it contains that will proliferate and provide the basis for tissues and organs of the shoot system are still largely undetermined at this time. It is only as the mitotic products of these initials are displaced away from the apical meristem that they take on their characteristic identities.