Behind the Hues of Green

Published in Lab times 01-2013.

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Electron micrographs of chloroplasts, the plant machinery that makes starch, against a backdrop of Arabidopsis seedlings

Besides fueling plants with their exceptional photosynthetic activity, chloroplasts can reversibly morph into other pigment-bearing structures with varied functions. But what directs such proteome reorganizations? The Jarvis group in Leicester enlighten us.

Tens of thousands of tourists flock southern Canada and New England in mid October each year to witness the fall foliage. The “leaf peepers” as they are called, gather in groups for a visual treat of colors – faded green, bright yellow, flaming orange, brilliant red and pretty purple – and herald the Indian Summer. It precedes that time of the year when days begin to get depressingly shorter and when the mercury drops sharply, forcing leaves off the trees to reveal their quivering skeletons. When the converse happens in spring, trees get back their lush greens and are swiftly adorned by intensely coloured flowers and fruits. Certainly, seasonal cues are reasons enough not only to drive exfoliation of trees but also to promote fruiting and trigger colour changes of their foliage.

Many of the colors of flora reflect the varied pigments made in tiny cellular compartments called plastids. Plastid morphology is an exciting field of plant biology given that the prototypical chloroplasts, green-pigment synthesizers of plant cells, are distinctively photosynthetic. Not just that, chloroplasts collect environmental cues to convert to other non-photosynthetic plastids viz. etioplasts in darkness, chromoplasts in fruits or gerontoplasts in aging leaves. Plastid development and transitions arise from changes in protein and pigment content within these compartments. When chloroplasts stock up on pigment carotenoids (chromoplasts) during fruiting for example, they paint flora with different hues and shades of red, orange and yellow colours. Such plasticity further, makes a compelling topic for research.

Now, what determines the protein composition (proteome) of plastids? Though plastids have their own protein synthesizing machinery, most of their proteins are nucleus-encoded and find their way in by a protein import process. Paul Jarvis, Professor of Plant Biology heads a lab at the University of Leicester, UK, that is focused on adding pieces to the ‘import’ puzzle. Their recent discovery of an important control mechanism of chloroplast protein import was published in Science (Science. vol.338(2):655-59), and it is hoped that their findings can be applied in agriculture from crop sustenance to fruit ripening.

An impressive start

Paul Jarvis was a postdoc at the Salk Institute in California when he identified the first chloroplast protein import mutant, plastid protein import 1 (ppi1) in Arabidopsis thaliana. The mutant lacks a then novel component of the translocon of the outer envelope of chloroplasts (TOC) and exhibits aberrant plastid and leaf development (Science. vol.282:100-103). “It was quite a breakthrough, and as it happens we eventually published that work in Science”, Paul recalls his prime in the year 1998. “It seemed such an interesting and important area of biology that I decided, when I first came to Leicester, to make it the major focus of the work in my laboratory”. With his strength in genetics, the Briton soon carved a niche in plant biology to address unknown aspects of chloroplast protein import.

Reaching out for suppressors

The TOC machinery is not one protein but a large complex of channel and receptor proteins that bind and permit cytosolic pre-proteins into the chloroplast; the ppi1 mutation affects one of the receptors. The most obvious thing to do for the geneticist hence was to rescue the import defects in the ppi1 mutant. In a “long-running project”, Paul mentored his first PhD student, Amy Baldwin in screening for second-site suppressors of ppi1 – they called one of these mutants sp1, for suppressor of ppi1 locus. Double mutant ppi1 sp1 plants exhibited recovery in chloroplast ultrastructure and protein import. Some years later, after much toiling with map-based cloning, Paul and his co-workers fished out the suppressor gene, SP1. Paul recollects this moment of Eureka with a sigh, “identifying the SP1 gene was the biggest breakthrough; the method we used is quite laborious and time-consuming, and to reach the end of that phase of the work and to be rewarded with such an exciting discovery was very fulfilling”.

Of course the ordeal did not stop there as the Jarvis group worked along different lines to decipher the nature and the role of SP1 in chloroplast development. It turned out that SP1 is a component of the ubiquitin proteasome system (UPS), a regulatory mechanism that destroys nonessential cellular proteins marked with a ‘ubiquitin’ tag and recycles them. Belonging to the family of E3 ligases, SP1 in fact confers substrate specificity to the UPS pathway and hence is a crucial determinant of its regulatory effects.

Now, having identified the fish, the botanists looked for the bait – the substrates. They found that the abundance of TOC proteins negatively correlated with that of SP1 and their hunch came true when a series of biochemical experiments uncovered receptor and channel components of the TOC machinery as SP1’s targets. SP1 at the outer surface of the chloroplast modifies the TOC with ubiquitin and facilitates its turnover by the UPS. This came about as a striking discovery since the UPS, a cytosolic pathway, was for the first time identified to have a role in the regulation of plastid proteins. As Paul puts it, “by targeting the import machinery, the cytosolic UPS can indirectly affect the interior – the nature of the proteins imported into the plastid by the TOC”.

Broader implications for the ligase

If SP1 targets the TOC, what then are the proteins downstream whose import is affected? Though Paul does not yet have a concrete answer to this question, he speculates that SP1 has a more generalized role in regulating protein trafficking mediated by the TOC. “The TOC machinery consists of many different receptor isoforms and functions in different import pathways – one very busy pathway that imports photosynthetic proteins as well as other pathways that transport non-photosynthetic or housekeeping proteins. SP1 serves to balance protein import by the TOC along these different pathways”.

Indeed, SP1 does not merely control a certain aspect of plastid development, but instead has a wider range of functions in plastid inter-conversions and senescence. The sp1 mutants fail to de-etiolate (the process of conversion of etioplasts to chloroplasts on exposure to light) efficiently and similarly, they display defective chloroplast to gerontoplast transitions delaying senescence. “SP1’s effect on TOC is not to turn it off but only to fine-tune its activity”, Paul summarizes.

Newer models newer aspirations

Conventionally, the lab’s favourite model organism has been the thale cress (Arabidopsis thaliana). “Arabidopsis has a relatively short generation time of about two months; second, it produces a lot of seed and hence, a lot of material for our experiments; and third, there’re plenty of resources out there making it the number one model system for plant science research”, elaborates Paul underscoring the merits of his dear plant. Because of the small size of its genome, Arabidopsis was the first plant to be selected for genome sequencing and all of its data are available on online resources such as The Arabidopsis Information Resource (TAIR). To top it all, generating transformants by Agrobacterium-based T-DNA insertions is a very easy task and knockouts for more-or-less any of Arabidopsis’ genes are accessible from stock centers worldwide.

However, the Jarvis lab is on the lookout for newer models to answer the questions that have surged in their recent paper. Owing to its crucial role in regulating chloroplast protein import, SP1 is a likely player in the chloroplast-to-chromoplast transition during fruit ripening. “By manipulating the activity of SP1, it may be possible to speed up or slow down fruit ripening”, Paul speculates. “We will use other species such as the tomato to analyze gene functions controlling transition from green chloroplasts to bright red chromoplasts in these fruits”. If they take the lead in this direction, Paul and colleagues wish to decode aspects of plastid development that can, ultimately, be manipulated to prolong shelf-life of fruits or accentuate ripening based on demand.

The setting

Towards the end of our tête-à-tête, the Botany professor makes a special mention of his lab and of Biotechnology and Biological Sciences Research Council (BBSRC), the two sole support systems in his avenues. “It is challenging to be part of plant science research for the main reason that unlike animal research, there are fewer options for funding and studies can take longer, but BBSRC appreciates the value of the work that we do”, he points out.

But whatever be the challenges, Paul hopes to translate his ideas into useful tools for agriculture as he walks back to his plant pots.

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