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

The Mechanism of Variegation in immutans Provides Insight into Chloroplast Biogenesis

The immutans (im) variegation mutant of Arabidopsis has green and white-sectored leaves due to the absence of fully functional plastid terminal oxidase (PTOX), a plastoquinol oxidase in thylakoid membranes. PTOX appears to be at the nexus of a growing number of biochemical pathways in the plastid, including carotenoid biosynthesis, PSI cyclic electron flow, and chlororespiration. During the early steps of chloroplast biogenesis, PTOX serves as an alternate electron sink and is a prime determinant of the redox poise of the developing photosynthetic apparatus. Whereas a lack of PTOX causes the formation of photooxidized plastids in the white sectors of im, compensating mechanisms allow the green sectors to escape the effects of the mutation. This manuscript provides an update on PTOX, the mechanism of im variegation, and findings about im compensatory mechanisms

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.</p

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

Chloroplast Photooxidation-Induced Transcriptome Reprogramming in Arabidopsis immutans White Leaf Sectors1[W][OA]

Arabidopsis (Arabidopsis thaliana) immutans (im) has green and white sectoring due to the action of a nuclear recessive gene, IMMUTANS. The green sectors contain normal-appearing chloroplasts, whereas the white sectors contain abnormal chloroplasts that lack colored carotenoids due to a defect in phytoene desaturase activity. Previous biochemical and molecular characterizations of the green leaf sectors revealed alterations suggestive of a source-sink relationship between the green and white sectors of im. In this study, we use an Affymetrix ATH1 oligoarray to further explore the nature of sink metabolism in im white tissues. We show that lack of colored carotenoids in the im white tissues elicits a differential response from a large number of genes involved in various cellular processes and stress responses. Gene expression patterns correlate with the repression of photosynthesis and photosynthesis-related processes in im white tissues, with an induction of Suc catabolism and transport, and with mitochondrial electron transport and fermentation. These results suggest that energy is derived via aerobic and anaerobic metabolism of imported sugar in im white tissues for growth and development. We also show that oxidative stress responses are largely induced in im white tissues; however, im green sectors develop additional energy-dissipating mechanisms that perhaps allow for the formation of green sectors. Furthermore, a comparison of the transcriptomes of im white and norflurazon-treated white leaf tissues reveals global as well as tissue-specific responses to photooxidation. We conclude that the differences in the mechanism of phytoene desaturase inhibition play an important role in differentiating these two white tissues

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

Expression of pathogen response genes.

<p>Real-time qPCR evaluation of pathogen-related genes involved in immune response in (A) non-infected and (B) infected plants at 4 DPI. The samples were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150983#pone.0150983.g006" target="_blank">Fig 6B</a>, and the genes are described in the text.</p

Tissue anatomy and plastid numbers.

<p>Leaves from 4- and 8-week-old Col-0 and <i>im</i> (white and green sectors) were fixed, stained and examined by light microscopy. (A, D, G) <i>im</i> white; (B, E, H) <i>im</i> green; (C, F, I) Col-0; (A, B, C) 1 month-old; (D–I) 2 month-old. (A-F) Sections were stained with toluidine blue and plastids were counted using Image J software (NCBI website); (J) the sections were 500 nm thick and approximately 150 cells were analyzed for each tissue-type. Asterisks indicate significant difference (t-test, p < 0.01). (G-I) Sections from 2-month-old leaf tissues were stained with Schiff’s reagent for starch. TEM images of representative plastids from fully-expanded <i>im</i> white (K) and wild type leaves (L).</p