In vivo three-dimensional imaging of plants with optical coherence microscopy.

Achieving the ability to non-destructively, non-invasively examine subsurface features of living multicellular organisms at a microscopic level is currently a challenge for biologists. Optical coherence microscopy (OCM) is a new photonics-based technology that can be used to address this challenge. OCM takes advantage of refractive properties of biological molecules to generate three-dimensional images that can be viewed with a computer. We describe new data processing techniques and a different visualization algorithm that substantially improve OCM images. We have applied OCM imaging, in conjunction with these improvements, to a variety of structures of plants, including leaves, flowers, ovules and germinating seeds, and describe the visualization of cellular and subcellular structures within intact plants. We present evidence, based on detailed examination of our OCM images, comparisons to classical plant anatomy studies, and current knowledge of light scattering by cells and their components, that we can distinguish nuclei, organelles and vacuoles. Detailed examination of vascular tissue, which contains cells with elaborate wall structure, shows that cell walls produce no significant OCM signal. These improvements to the visualization process, together with the powerful non-invasive, non-destructive aspects of the technology, will broaden the application of OCM to questions in studies of plants as well as animals.

The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots.

Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions.

Optical coherence microscopy. A technology for rapid, in vivo, non-destructive visualization of plants and plant cells.

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 microm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 x 5 x 10 microm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.

Expression of a delta 9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of omega 5 anacardic acids found in pest-resistant geranium (Pelargonium xhortorum).

Anacardic acids, a class of secondary compounds derived from fatty acids, are found in a variety of dicotyledonous families. Pest resistance (e.g., spider mites and aphids) in Pelargonium xhortorum (geranium) is associated with high levels (approximately 81%) of unsaturated 22:1 omega 5 and 24:1 omega 5 anacardic acids in the glandular trichome exudate. A single dominant locus controls the production of these omega 5 anacardic acids, which arise from novel 16:1 delta 11 and 18:1 delta 13 fatty acids. We describe the isolation and characterization of a cDNA encoding a unique delta 9 14:0-acyl carrier protein fatty acid desaturase. Several lines of evidence indicated that expression of this desaturase leads to the production of the omega 5 anacardic acids involved in pest resistance. First, its expression was found in pest-resistant, but not suspectible, plants and its expression followed the production of the omega 5 anacardic acids in segregating populations. Second, its expression and the occurrence of the novel 16:1 delta 11 and 18:1 delta 13 fatty acids and the omega 5 anacardic acids were specific to tall glandular trichomes. Third, assays of the recombinant protein demonstrated that this desaturase produced the 14:1 delta 9 fatty acid precursor to the novel 16:1 delta 11 and 18:1 delta 13 fatty acids. Based on our genetic and biochemical studies, we conclude that expression of this delta 9 14:0-ACP desaturase gene is required for the production of omega 5 anacardic acids that have been shown to be necessary for pest resistance in geranium.

Isolation of an Arabidopsis homologue of the maize homeobox Knotted-1 gene.

The shoot apical meristem (SAM) is responsible for forming most of the above-ground portion of the plant. We sought to isolate regulatory genes expressed in the Arabidopsis SAM by screening a Brassica oleracea (cauliflower) meristem cDNA library with the homeobox fragment from the maize Knotted-1 (Kn1) gene. We isolated and characterized the corresponding clone, Merihb1, from Arabidopsis. Analysis shows that the predicted MERIHB1 protein exhibits strong homology to KN1 and RS1 from maize, SBH1 from soybean, and KNAT1 and KNAT2 from Arabidopsis. Merihb1 is highly expressed in mRNA from cauliflower meristems and also accumulates in stem and flower mRNA. Based on the similarity of the Merihb1 and Kn1 sequences, expression patterns, and in situ hybridizations, we suggest that Merihb1 represents an Arabidopsis homologue of the maize Kn1 gene.

A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis.

The KNOTTED class of plant genes encodes homeodomain proteins. These genes have been found in all plant species where they have been sought and, where examined, show expression patterns that suggest they play an important role in shoot meristem function. Until now, all mutant phenotypes associated with these genes have been due to gain-of-function mutations, making it difficult to deduce their wild-type function. Here we present evidence that the Arabidopsis SHOOT-MERISTEMLESS (STM) gene, required for shoot apical meristem formation during embryogenesis, encodes a class I KNOTTED-like protein. We also describe the expression pattern of this gene in the wild-type plant. To our knowledge, STM is the first gene shown to mark a specific pattern element in the developing plant embryo both phenotypically and molecularly.

The forever young gene encodes an oxidoreductase required for proper development of the Arabidopsis vegetative shoot apex.

In plant development, leaf primordia are formed on the flanks of the shoot apical meristem in a highly predictable pattern. The cells that give rise to a primordium are sequestered from the apical meristem. Maintenance of the meristem requires that these cells be replaced by the addition of new cells. Despite the central role of these activities in development, the mechanism controlling and coordinating them is poorly understood. These processes have been characterized in the Arabidopsis mutant forever young (fey). The fey mutation results in a disruption of leaf positioning and meristem maintenance. The predicted FEY protein shares significant homology to a nodulin and limited homology to various reductases. It is proposed that FEY plays a role in communication in the shoot apex through the modification of a factor regulating meristem development.

Normal and Abnormal Development in the Arabidopsis Vegetative Shoot Apex.

Vegetative development in the Arabidopsis shoot apex follows both sequential and repetitive steps. Early in development, the young vegetative meristem is flat and has a rectangular shape with bilateral symmetry. The first pair of leaf primordia is radially symmetrical and is initiated on opposite sides of the meristem. As development proceeds, the meristem changes first to a bilaterally symmetrical trapezoid and then to a radially symmetrical dome. Vegetative development from the domed meristem continues as leaves are initiated in a repetitive manner. Abnormal development of the vegetative shoot apex is described for a number of mutants. The mutants we describe fall into at least three classes: (1) lesions in the shoot apex that do not show an apparent alteration in the shoot apical meristem, (2) lesions in the apical meristem that also (directly or indirectly) alter leaf primordia, and (3) lesions in the apical meristem that alter meristem size and leaf number but not leaf morphology. These mutations provide tools both to genetically analyze vegetative development of the shoot apex and to learn how vegetative development influences floral development.

Molecular cloning and characterization of genes expressed in shoot apical meristems.

The above-ground portion of a plant develops from the shoot apical meristem. An abundant source of apical meristems was obtained from cauliflower heads. Meristematic cDNAs were identified by differential screening and used to isolate corresponding Arabidopsis thaliana genes. Transcriptional promoters from Arabidopsis clones were fused to the beta-glucuronidase (GUS) reporter gene and introduced into plants, and GUS expression was used to analyze temporal and spatial regulation of the promoters. One promoter (meri-5) directed GUS expression in the meristematic dome and not the surrounding leaf primordia. The meri-5 promoter also directed GUS expression at branching points in the shoot and root. A second meristematic gene was found to be a histone (H3) gene. The H3 promoter was isolated and fused to GUS. Expression of the H3-GUS fusion in transgenic tobacco showed preferential expression in the peripheral zone and a lack of noticeable staining in the central zone.

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.

Regulation of chlorophyll and Rubisco levels in embryonic cotyledons of Phaseolus vulgaris.

While deep within the maternal tissues (pods and testa), cotyledons of the bean (Phaseolus vulgaris L.) green and the plastids differentiate as chloroplasts. At the time of seed maturation the chloroplasts dedifferentiate and the green color is lost. We have used Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll to study chloroembryo development. Chlorophyll levels and Rubisco activity increase early in embryonic development then decline as the cotyledons enter the maturation phase. Rubisco accumulation follows a strong temporal pattern over the course of embryo development, and furthermore, occurs in total darkness. Therefore, accumulation of Rubisco during embryogenesis may occur in response to developmental signals. In embryos developed in total darkness, Rubisco accumulation was uncoupled from chlorophyll accumulation. Exposure of isolated cotyledons to abscisic acid (ABA) resulted in loss of chlorophyll and decline in Rubisco levels comparable to those seen in normal embryogenesis. This indicates that the decline in Rubisco in chloroembryos in vivo results from factors such as ABA that signal the onset of maturation. The results show that ABA not only enhances the accumulation of some proteins (e.g. storage proteins), but also depresses the accumulation of others during embryogeny.

Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases.

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3' nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture.