A study of chloroplast biogenesis within variegated mutants of Arabidopsis

immutans(im) is a recessive Arabidopsis mutant that is involved in chloroplast biogenesis. In the absence of IMMUTANS (IM) green and white leaf sectors form. This thesis is concerned with the development of these green and white sectors and the methods used to investigate the mechanism of their formation. IM resides in the chloroplast thylakoid membrane and has been implicated in the adjustment of electron flux through the photosynthetic electron transport chain. Without IM white sector formation occurs due to the inability of im to produce colored carotenoids. It is still a mystery as to how green sectors are able to form in the absence of IM. This thesis presents im as a developmental chloroplast defect and discusses the need for IM very early in plastid development. The requirement for IM most likely starts within progenitor plastids known as proplastids

PDS activity acts as a rheostat of retrograde signaling during early chloroplast biogenesis

Chloroplasts are crucial for the process of photosynthesis, as well as for developmental and environmental sensing. One of the important mechanisms of sensing is retrograde (plastid-to-nucleus) signaling, whereby the state of the chloroplast is signaled to the nucleus, resulting in alterations in gene expression for chloroplast proteins, usually at the transcriptional level. Retrograde signaling was early studied in carotenoid-deficient plants that contain, upon exposure to high light, photooxidized plastids that arise because of an inability to quench ROS produced during the light reactions of photosynthesis. Phytoene desaturase (PDS) is required for one of the early steps of the carotenogenic pathway, and impaired PDS activity during early chloroplast biogenesis results in a highly reduced plastoquinone pool (high excitation pressure), accumulation of the colorless C40 intermediate, phytoene and white photooxidized plastids. Here, we discuss results from global transcript profiling of white leaf tissues of Arabidopsis that are blocked at the PDS step in three different ways—two by mutation (immutans & pds3) and one by inhibitor treatment (norflurazon). We show that the molecular phenotypes of the three tissues bear many similarities, but that there are also significant tissue-specific differences. We propose that PDS acts as a rheostat of excitation pressure-mediated retrograde signaling during chloroplast development and speculate that whether the rheostat is set high (as in pds3 and NF-treated seedlings), intermediate (as in im) or low (as in WT) is a crucial determinant of the suite of genes that is expressed during chloroplast biogenesis

Impaired Chloroplast Biogenesis in Immutans, an Arabidopsis Variegation Mutant, Modifies Developmental Programming, Cell Wall Composition and Resistance to Pseudomonas syringae.

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions

Monosaccharide composition (mol%) of cell wall from immutans and <i>Col-0</i> rosette leaves.

<p>Monosaccharide composition (mol%) of cell wall from immutans and <i>Col-0</i> rosette leaves.</p

Evans blue exclusion test.

<p>(A) Leaves of <i>im</i> and Col-0 were infiltrated with a <i>P</i>. <i>syringae</i> cell culture at a density of 10⁴ colony forming units (cfu), and bacterial growth was monitored daily for 4 days after infection (DPI). (B) Leaves were detached from 7 week-old Col-0 and <i>im</i> and stained with Evans blue. In some experiments, detached wild type leaves were infiltrated with a <i>P</i>. <i>syringae</i> cell culture (density of 10⁴ cfu) prior to staining. In the Evans blue exclusion test, living cells exclude the dye, while dead cells take it up.</p

Composition of cell wall polymers.

<p>(A) Lignin and cellulose contents in the cell walls of Col-0 and <i>im</i> (white and green sectors). The contents were determined on a per mg basis of cell wall extracts. (B) Callose accumulation in Col-0 and <i>im</i> (white and green sectors) before and after infection with <i>P</i>. <i>syringae</i>. Expanded leaves from 7 week-old plants were cleared with ethanol and stained with aniline blue. The images were captured via UV fluoresecence microscopy. Bars = 200 μm.</p