Delayed greening in theobroma cacao L 2009
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This document summarizes research on delayed greening in Theobroma cacao leaves. Delayed greening refers to leaves initially being yellow and lacking chlorophyll, before developing green color later in maturation. The document reports that leaf growth precedes chlorophyll accumulation in T. cacao. Photosynthesis capacity and efficiency develop later still, in the third phase of leaf maturation. Experiments with leaf discs cultured in vitro show that growth continues with sugar supplementation, but chlorophyll accumulation stops, indicating greening is regulated separately from growth. The aim of the research is to further study chloroplast development and signaling using T. cacao as a model plant.
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Delayed greening in theobroma cacao L 2009
- 1. 8th September 2009 MitgliedderHelmholtz-Gemeinschaft Delayed greening in Theobroma cacao L. Leaf and photosynthesis development | Andrzej Stefan Czech
- 2. 8th September 2009 Slide 2 INTRODUCTION
- 3. 8th September 2009 Slide 3 What is normal greening Young leaf is green from the beginnig Young leaves look like mature ones, but are smaller Chloroplasts become mature early in the development Leaf performs photosythesis very early in the development
- 4. 8th September 2009 Slide 4 What is normal greening Čatský and Šesták 1996
- 5. 8th September 2009 Slide 5 What is delayed greening Present in 33% of 250 tested tropical species, shade plants Low chlorophyll content at the beginning of the leaf development Little chloroplasts Leaves yellow, thin, transparent, floppy No cuticle
- 6. 8th September 2009 Slide 6 Delayed greening Young leaf Mature leaf intercellular air space
- 7. 8th September 2009 Slide 7 Etioplast Mature chloroplast Young delayed greening chloroplast Baker, Hardwick, Jones 1975 Baker, Hardwick, Jones 1975 von Sengbusch 2003 PS I PS I granum thylakoid Prolamelar bodies mainly with POR protein only Chloroplast development
- 8. 8th September 2009 Slide 8 Ecological meaning of delayed greening Masking (defense against herbivores) Economy (time distribution of energy consuming processes) Photoprotection (defense against photosensibilization)
- 9. 8th September 2009 Slide 9 Aim To make a model of development of T. cacao leaves for further research Motivation T. cacao can be a model plant for chloroplast development due to tiny chloroplast at the beginning of leaf development. It may be a better model than etioplasts.
- 10. 8th September 2009 Slide 10 RESULTS
- 11. 8th September 2009 Slide 11 Leaf growth and chlorophyll accumulation Growth preceeds chlorophyll accumulation Days after reaching 1.5 cm in length 0 10 20 30 40 RelativeGrowthRate[%d-1] 0 20 40 60 80 100 Chlorophyll[molm-2] 0 100 200 300 400 500 600 %Amax[%] 0 20 40 60 80 100 120 Relative Growth Rate Chlorophyll %Amax
- 12. 8th September 2009 Slide 13 Growth of delayed greening versus normal
- 13. 8th September 2009 Slide 14 Leaf growth versus chlorophyll accumulation Chl [mol m-2] 0 100 200 300 400 500 600 RelativeGrowthRate[%d-1] 0 20 40 60 80 100 greenhouse growth chamber VIVIIIIII There seems to be a correlation between growth and chlorophyll accumulation in phases II and III
- 14. 8th September 2009 Slide 15 Cell size versus chlorophyll accumulation Chl [mol m-2] 0 100 200 300 400 500 600 Epidermalcellsize[m2] 0 100 200 300 400 500 VIVIIIIII Rapid growth in the first phase is due to cell division, in phases II and III only cell expansion happens.
- 15. 8th September 2009 Slide 16 Cell growth of delayed greening versus normal
- 16. 8th September 2009 Slide 17 Spatial leaf growth Growth pattern different from other dicots Arabidopsis thaliana Nicotiana tabacum Theobroma cacao High growth Low growth Wiese et al, 2007 Walter et al, 2005
- 17. 8th September 2009 Slide 18 Photosynthesis development Chl [mol m-2] 0 100 200 300 400 500 600 Photosynthesis [molCO2m-2s-1] -2 -1 0 1 2 3 4 VIVIIIIII Development of CO2 assimilation capacity happens in phase III, when most of the growth is already accomplished.
- 18. 8th September 2009 Slide 19 Photosystem II efficiency versus chlorophyll content Chl [mol m-2] 0 100 200 300 400 500 600 PhotosystemIIefficiency 0.65 0.70 0.75 0.80 0.85 VIVIIIIII Increase in photosystem II efficiency happens in phase III, when most of the growth is already accomplished. Bassi, 2009 Bassi, 2009
- 19. 8th September 2009 Slide 20 NPQ versus chlorophyll content VIVIIIIII Chl [mol m-2] 0 100 200 300 400 500 600 NPQ 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 y = 0.0019 x + 1.4317 R2 = 0.7639 Protective mechanisms are developed together with chlorophyll accumulation.
- 20. 8th September 2009 Slide 21 VIVIIIIII Chl [mol m-2] 0 100 200 300 400 500 600 Carbohydrates [molg-1FW] 0 10 20 30 40 50 60 70 glucose fructose sucrose starch Carbohydrates versus chlorophyll content Although in phase IV leaf is self-sufficient, it starts to function as active sugars exporter in phase V.
- 21. 8th September 2009 Slide 22 Photosynthesis development of delayed greening versus normal
- 22. 8th September 2009 Slide 23 SUMMARY
- 23. 8th September 2009 Slide 24 LAST (NOT YET PUBLISHED) RESEARCH
- 24. 8th September 2009 Slide 25 In vitro analyses of chloroplast development Aim: to establish a setup allowing simple and effective modification of growth conditions, to test the influence of sugar, hormones, light, etc. on chloroplast development Approach: leaf discs, cut from growing or maturing leaves, grown on liquid (or solid) media (Hoagland or MS)
- 25. 8th September 2009 Slide 26 Growth of leaf discs control Hoagland Hoagland +1% sucrose Hoagland +3% sucrose Hoagland +0.53% mannitol Hoagland +1.6% mannitol maturing leaf 160 µmol m-2 Chl growing leaf 100 µmol m-2 Chl Leaf discs are able to grow if sugars are supplied. RGR[%d-1] 7daysaverage 0 2 4 6 8 10 12 14 16 18 20 maturing leaf growing leaf
- 26. 8th September 2009 Slide 27 Growth of leaf discs (dry weight) Leaf discs are growing, not only swelling. Treatment Hoagland Hoagland + 1% sucrose Hoagland + 3% sucrose Hoagland + 0.53% mannitol Hoagland + 1.6% mannitol DW[mgperdisc] 0 1 2 3 4 5 6 7 maturating leaf growing leaf
- 27. 8th September 2009 Slide 28 Chlorophyll accumulation in leaf discs Treatment control Hoagland Hoagland + 1% sucrose Hoagland + 3% sucrose Hoagland + 0.53% mannitol Hoagland + 1.6% mannitol Chl[molm-2] -50 0 50 100 150 200 maturing leaf growing leaf
- 28. 8th September 2009 Slide 29 Why chlorophyll accumulation in leaf discs is stopped while growth not? Maybe growth is internally controlled by cells (e.g. programmed at the very beginning of leaf development) while greening is regulated by plant (via phytohormons) or restricted by nutrient availability. Further experiments in progress.
- 29. 8th September 2009 Slide 30 Future perspectives Search for signal(s) for „greening” (chloroplast development). Comparison of normally developing leaves with leaves where greening is artificially stopped. Changes in the thylakoid membrane structure and functionality of photosystem II.
- 30. 8th September 2009 Slide 31 Acknowledgements Forschungszentrum Jülich – Jagiellonian University scholarschip Ulrich Schurr, ICG-III, Forschungszentrum Jülich, Germany Shizue Matsubara, ICG-III, Forschungszentrum Jülich, Germany Kazimierz Strzałka, ZFIBR, WBBiBt, Jagiellonian University, Kraków, Poland Thank you for your attention.
Editor's Notes
- Thank you for the opportunity to present the work I have been doing in the Forschungszentrum in the last two years. My research focus on delayed greening in Theobroma cacao.
- First I would like to introduce you a little to this topic. I would like to start with the explanation of normal greening, that we can experience looking at the trees outside, behind the window.
- So what is this normal greening? First, when we look at a young leaves we have to notice, that they are green. Maybe not as green as the mature ones but definitely green. And they look like the mature ones with the exception that they are smaller. If we look a bit closer, will notice, that the chloroplasts maturate very early in the leaf development. And also early they start to live on their own – in the meaning, that they perform photosynthesis and don’t live on the cost of the rest of the plant.
- On this graph you can see, how the normally greening leaf develop. After the leaf initiation it is growing until it reaches mature size there. The growth is happening first due to cell division, and than due to cell expansion. And very early in the leaf life photosynthesis, here presented as CO2 exchange rate, becomes positive and reaches its maximal level long before the leaf is fully expanded.
- With the delayed greening the situation is different. This phenomenon can be found in many tropical plant species, which grow in shade in the understory. The typical symptoms of delayed greening that you can see on the picture, are very low amount of chlorophyll in the leaf and very tiny chloroplasts. Leaves are usually yellow, thin, transparent and they hang down in vertical position. They also do not have much of cuticle that covers mature leaves. And as soon as they reach mature size, they become horizontal, tough, green and fully functional.
- Here you have two cross-sections of young and mature leaf. In the young leaf there is not much space between cells, so it makes gas exchange difficult and in the mature leaf there are large air spaces that allow easy gas exchange that is essential for effective photosynthesis.
- In current researches most of investigations in chloroplast formation are done either on plant embryos or on etioplasts formed in the darkness that are transformed into chloroplasts upon the light. But delayed greening leaves have chloroplasts that resemble mature chloroplasts having the same structure of photosynthetic membranes - thylakoids forming something like a granum, that is a very densly stacked membrane system. The difference is, that they are much, much smaller and very simple structured. Etioplast have very regular structure of prolamelar bodies and also the composition of the proteins is different: mainly POR proteins that bind protochlorophyllide – a chlorophyll precursor, while young delayed greening chloroplasts have the composition similar to that of the mature chloroplasts with photosystems I and II.
- There are a few explanations why delayed greening is happening. First suggest masking as the defence against herbivores. That means, that if leaf is not green but yellow it does not look like a leaf but like a flower that usually contains less proteins hence not as attractive to the insect. Second explanation is the economy. Building of the leaf structure and the photosynthetic apparatus are a very energy consuming processes so if you distribute them in time you will have lower requirements for sugars and nutrients in one time. The last explenation suggests that if you have chlorophyll you need all the protective mechanisms that defend against photosensibilization. These plants live in shade, but they can be exposed to sudden light flecks, so it would be better to not to have chlorophyll until they can utilise light energy absorbed by chlorophyll to drive photosynthetic electron transport.
- The aim of my research was to make a model of development of the leaves for the further research and especially I am interested in the development of chloroplasts and photosynthesis that are tightly connected.
- Now, I will switch to results of my work. The part I will present you now is recently accepted in Functional Plant Biology.
- So, how different is the delayed greening from normal greening?On this slide you can see how the development of delayed greening leaves is happening in time. In the beginning growth is very high and decreases as the leaf area increases. But for a few days there is no accumulation of chlorophyll in young leaves. And only after 5 or 6 days it begins to increase and continues to increase almost linearly even when the growth is already accomplished. Later, only a minor increase in chlorophyll content can be seen.
- If we compare than development of delayed greening with normal ones we can say, that leaf expansion is happening faster in the delayed greening leaves.
- If we plot growth against chlorophyll content we can see, that there is a clear correlation between these two parameters. And this correlation is independent from the conditions in which plants were grown. White dots represent plants grown in strictly controlled, optimal conditions and the black ones represent plants from greenhouse where light was highly variable, as well as temperature and humidity. But this two data series still fall in the same pattern. Because of this clear correlation and of the linear increase in chlorophyll content I will present all the next data on the chlorophyll content basis.
- If we will look at the cell size we see that in the first phase there is no increase in size, although growth is very high in this phase. That means that mostly cell divisions are happening here. Than we have a phase of rapid increase and third phase of slower increase. So, here leaf expansion is mainly accomplished due to cell expansion.
- In the comparison to the normal greening leaves we see, that the patter is similar, only time scale is shorter here.
- If we look at the spatial distribution of growth, we can see, that T.cacao grow homogenously on the whole area of the leaf lamina, while many other “normal” dicots have a pronounced base tip gradient. Base of the leaf is expanding, while tip is undergoing maturation only.
- If we will have a look at the photosynthesis we can see that it becomes positive in phase III when the leaf is almost fully expanded and it reaches maximal value somewhere in phase V that is long lime, after leaf is fully expanded. Most of the photosynthesis development is happening in phase III.
- If we measure photosystem II efficiency, we can see that in first 3 phases an increase of fluorescence is happening, and in last two phases there is little increase, although, chlorophyll content almost doubles in this time. We can explain it by the fact that first reaction centre is developed and later the light harvesting antenna.
- If we look at the carbohydrates levels during the leaf development we can see a quite high levels of glucose and fructose in the beginning, that are even increasing up to phase IV and than a decrease in phase V when leaves are mature. They represent the metabolic activity of the leaf. Sucrose which is the transport sugar, is high in the beginning and decreases in the phase III when there is a huge demand for sugar because of production of photosynthetic apparatus, which is a very energy consuming process. Than it increases in phase IV together with starch, the storage material and this increase can be explained by the fact, that leaf becomes self sufficient in this phase and can produce its own sugars due to photosynthesis, and do not have to import them from the rest of the plant. in the beginning growing leaves import sugars and later they accumulate and export more than they consume.
- If we compare photosynthesis of normal greening leaves with delayed greening leaves, we can see, that maximal photosynthesis of the last ones is reached much later in the leaf lifetime.
- Just to summarise this what I said before. Delayed greening leaves grow faster reaching the final size faster than the normal leaves. They show the similar pattern: first cell divisions and then cell expansion. But in contrast to normal greening leaves they need for a longer time to depend on imported sugars and reach the maximal photosynthesis much later in time. This gives us much more time to investigate the development of chloroplasts and photosynthetic membranes.
- Now, a short overview on my most recent research and some outlook.
- I am currently doing an in vitro analyses to test the influence of factors like sugars, phytohormones, light and other on the development of chloroplasts. For this I am using leaf discs, cut from growing or maturing leaves, that are growing on liquid or solid media in controlled and reproducible conditions.