Carotenoid biosynthesis in plants

CAROTENOIDS: LIFE SAVERS FOR PLANTS AND PEOPLE

Every early autumn, I take a car and drive away from Manhattan to the New Jersey suburbs. I go away from concrete jungles to watch a beauty of wilderness to get some peace in my mind. Each early September a green carpet of leaves turns into a terrific painting of yellow, orange, and red colors of every tone and intensity, inspiring poets to write poems, directors to shoot movies and biochemists like me to go away from the heart of the city to enjoy a scenic view.

I spent several years of my life studying the nature of these cheerful leaf colors. Autumn trees look so vibrant because their leaves accumulate pigments called carotenoids. Similar colors of fruits and vegetables, such as carrots, peppers, oranges, squash, melon or corn, are also caused by those bright pigments.

There are several reasons plants make carotenoids. Indeed, sometimes they do it to attract people and please them: colored fruits look nice to us or other animals and convince us to eat them. Once eaten, fruit seeds eventually make it to the outside world and fall in soil far away from their parental plants. A classic barter case, invented by nature long time ago: animals enjoy tasty fruits, and in exchange serve as seed distributors helping plants to take over the world.

Protective shields

Attracting seed distributors is not the only purpose of carotenoids. The main purpose of these pigments in leaves is to protect plants from sun damage. Green organisms are different from us. Plants do not need any organic food, but instead, they can make food out of air and sun light. Sun is a source of life for plants. Alive factories use a green molecule called chlorophyll to utilize light photons as free energy donors and synthesize sugars out of water and carbon dioxide. Sugars build the body of plants and eventually become food for all plant-eating living organisms. Plants are living preserves of sun energy for all of us. But the sun is not always nice to plants. They have to protect the gentle photo-synthetic systems from too much harmful light energy; and for that they use the help of molecular carotenoid shields. Carotenoids absorb additional excited light photons and either transfer them to chlorophyll, or if the photons are in excess, prevent a leaf from being burnt alive. Those are carotenoids we see in autumn leaves: usually masked during the summer by chlorophyll, they become visible when green molecules are disassembled and plants are getting ready for a winter rest.

Vitamin A

There are plenty of different plant carotenoid molecules, colors of which vary from none to yellow, orange or red. You might know one of them – beta-carotene, which accumulates in carrots, corn and other bright-colored or deep-green vegetables. We consume beta-carotene from plant foods and convert it to vitamin A. Remember your parents, trying to convince you to eat more vegetables? I believe there is a classic tradition, transferred from parents to children, to torture their kids with some steamed broccoli and carrots! And one of the great benefits of this tradition is the support of eye health. Vitamin A, together with other carotenoids such as lutein and zeaxanthin, play important role in our eye development and protection, and eating a lot of colored fruits and greens serves better and healthier eyes. The role of beta-carotene is so important, that in many parts of Africa millions of children suffer from eye diseases and blindness simply because their major food is white rice, and they do not have an access to those greens and vegetables.

Another tradition – eating fresh salads with dressing – also has an important underlying reason: carotenoids are fat-soluble, and dressings such as olive oil help better digestion. Carotenoids from food protect us from highly active molecules such as oxygen or sun ultraviolet, same way they do it in plants, absorbing harmful compounds and preventing aging and cancer. Not only carotenoids are protective shields for plants, they are shields for us! Those colored pigments are important for human health throughout the whole life.

Carotenoids: how are they made?

Plant molecular biologists have been studying carotenoid biosynthesis for over than 20 years. They discovered that it takes no less than 10 specific proteins to make different carotenoids in plants. Carotenoid biosynthesis begins with the production of a colorless carotenoid called phytoene. Then phytoene undergoes step-by-step conversions, each one supported by a specific protein, giving a variety of different colored carotenoids, some of which you can see on a picture below. Such a sequence of conversions is called a biosynthetic pathway.

The first step – making phytoene – is the most important step in all the pathway of carotenoid transformations. Without phytoene, the production of other carotenoids is not possible. Phytoene is made out of small precursor molecules, called geranyl geranyl pyrophosphate, by an enzyme called Phytoene Synthase, or PSY. In leaves, where carotenoids assist photosynthesis, PSY is always present to provide phytoene necessary to make other carotenoids and protect leaves. However, in other parts of plants, such as roots or fruits, PSY might be present or not, depending on a plant species. Colored fruits of tomatoes, oranges, or yellow corn all have PSY in their seed tissues. Color of fruits depends on the carotenoid accumulated, and is regulated by enzymes involved in both production and further modification of a carotenoid molecule. So, during biosynthetic pathway phytoene is eventually converted to lycopene, which gives red color to tomatoes, or alfa- or beta-carotenes that give deep yellow or orange color to corn, carrots, melon or squash. Uncolored seeds and fruits, such as white rice or white corn, lack carotenoids due to a simple reason: there is no PSY in them to provide phytoene to start the pathway.

Carotenoid biosynthesis research helped breeders to create plants with different amount of pigments. The genetic code for carotenoid producing proteins can be tracked in the DNA of the plant, so the composition and amount of carotenoids can be predicted just based on such DNA analysis. The analysis saves time for breeders, who can predict the features of the plant in its embryo, skipping the long waiting period the plant takes to mature. Predicting of carotenoid amount helps to breed plants that have high amount of vitamin A or other healthy carotenoids like lycopene, attractive color, or strong plants that are better protected from environmental stress. Such plants can grow in regions that traditionally are not good for agriculture.

However, not every plant can be easily bred to change carotenoid amounts. Traditional breeding can modify or eliminate certain genes from genome, but sometimes the process takes thousands and thousands of years. For example, a well-known and popular yellow corn is a result of a lucky accident: several thousand years ago a Mexican farmer noticed a yellow plant in a huge field of white corn and thought it was pretty. We are thankful for that farmer, who picked this vitamin A-rich variety for us, and now we know, that the ability of maize to produce carotenoids in seeds happened due to a very rare mutation.

Golden Rice

As we know, there are no carotenoids in seeds of rice. After years of breeding we realize that there is not a slight chance that rice is able to turn orange by itself. As we know now from DNA analysis, rice does not have a working PSY (and as a result – no phytoene to start the pathway with) in its grains. It would be a great thing to have rice with beta-carotene; it would help African nations who rely upon it as a major staple to avoid vitamin A deficiency. Nowadays, we have modern methods to solve this problem. Plant genetic engineers used tools of molecular biology and cloning to introduce PSY from corn into rice seeds. It occurred that for rice has PSY in leaves, but for some reason rice seeds lack PSY and at the same time have all the enzymes to convert lycopene into beta-carotene and later carotenoids. Corn PSY, inserted into plants together with another protein assisting conversion of phytoene to lycopene, were produced together in rice seeds, and able to successfully complete the carotenoid biosynthetic pathway making beta-carotene. This deep orange rice received a name of “Golden Rice”. Seeds of Golden Rice have a lot of beta-carotene, and in several years after thorough testing for safety Golden Rice will be donated to developing countries of Africa for free distribution.

Why several PSY?

There are still many questions on carotenoid biosynthesis in plants. Recent success in full plant genome sequencing discovered, that there are not only one, but two or three sub-types of almost each carotenoid enzyme. For example, there are three (!)  PSYs in corn – one for leaves, one for fruits, and one for roots. The reason plants are not happy with only one version of the enzyme and need several specific sub-types is yet unknown; however, we showed in our lab that it might be related to the shape of the enzyme, thus affecting its location in a cell, and ability to work or connect to other enzymes of the pathway. Check the cover image of this web-site: you can see one of the versions of PSY enzyme under the microscope in the corn leaf cell. We attached a green fluorescent marker  to the enzyme, so using a fluorescent light we can detect our PSY. The red spheres are chloroplasts – that is how they look like under fluorescence,  and the green diamons around them are PSY enzyme.  A grey image to the right is showing how the cells look like under a light microscope with no fluorescence – it would be impossible to detect the enzyme without our techniques.

We hope that our further research will improve the knowledge on carotenoid biosynthesis and help more efficient breeding or designing new plants with better nutritional features or resistant to coming climate change.

ADDITIONAL READING:

1.  Shumskaya, M. and Wurtzel, E.T. (2013) The carotenoid biosynthetic pathway: thinking in all dimensions.  Plant Science, 208: 58–63 (invited review). Download Full Text (Plant Science 2013)
2. Shumskaya M., Bradbury L.M.T., Monaco R., and Wurtzel E.T. (2012) Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity. Plant Cell, 24 (9): 3725-3741. Download Full Text (Plant Cell 2012)