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Synthetic engineering of membrane transport to increase photosynthesis and water use efficiency

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Universitat de Barcelona. Departament de Biologia Vegetal

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[eng]  The rapid increase on world’s population is threatening food security, mainly in developing countries, where poverty and hunger are already a big concern.  However, a growth in agriculture generates an overall economic growth and leads to a poverty reduction. The aim of this thesis was to develop synthetic biological solutions that might aid in increasing photosynthetic yields and water use efficiency of plants, thereby improving crop yields.

Two strategies were developed as a possible ways to manipulate the plant in order  to get an increase on photosynthesis and water use efficiency (WUE). The first was through the engineering of a carbon concentrating mechanism (CCM) in a C3 plant, in order to reduce the oxygenase activity of Rubisco and thus increasing photosynthesis. The second was through exploring the possibilities of changing stomatal kinetics in order to put in concordance the stomatal conductance with the mesophyll demand for CO2; thus not only the possibility of increasing photosynthesis but WUE.

In order to engineer a CCM into a C3 plant it was elaborated an artificial transport system in the inner envelope of the chloroplasts that could be used to increase the levels  of  HCO3-­‐  in  the  chloroplasts  stroma.  To  this  end,  the  light  driven  pump halorhodopsin from Natronomonas pharaonis (NpHR) was chosen and targeted to the inner envelope of the chloroplast with the aim to establish a Cl-­‐ gradient across the chloroplasts inner envelope. The human anion exchanger, AE1, was also targeted and introduced into the inner envelope of the chloroplasts, to allow an exchange of Cl-­‐ per HCO3-­‐, thus powering an increase in HCO3-­‐ in the chloroplast stroma. A third protein, bCMO1 was also incorporated to the system in order to provide necessary retinal for the proper activity of NpHR. Transgenic plants expressing the artificial membrane transport system were generated, however no increase on photosynthesis could be observed on the transformed plants. It might be necessary to create another synthetic system designed to retain the CO2 where the Rubisco is localised, in order for Rubisco to be able to fix it.

As possibilities to change stomatal kinetics two different approaches were studied. The first one was to manipulate the stomatal kinetics through the manipulation of the Kin channels V1/2. The second one was to manipulate the stomatal kinetics through the addition of the external pump NpHR. Both approaches were simulated on the guard cell model OnGuard. Out from the model predictions it was observed
 
that both approaches were able to manipulate the stomatal kinetics. By  manipulating Kin channels V1/2 to more positive values related to the control, the stomata opened faster, however it remained opened during the night. In contrast, when manipulating Kin channels V1/2 to more negative values, the stomata didn’t show much variation between the day and night, thus remaining almost closed during the whole day cycle. Surprisingly, the model predicted that by adding the NpHR in the plasma membrane of the guard cell, the stomata opened faster under a light stimuli and it also closed faster under a dark stimuli. Thus, presenting behaviour that could become to an increase on plant photosynthesis and WUE if reproducible in vivo. After a complex cloning work it was possible to create transgenic plants comprising all the characteristics previously reproduced into the model. These characteristics were from both, the Kin approach, on which the KAT1 channel was the selected one in order to be modified, and the NpHR approach. Experiments on gas exchange measurements should be done in order to prove if the generated plants have an increase on photosynthesis and WUE, which hasn’t been demonstrated yet.

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[cat]  El ràpid increment de la població mundial, sobretot als països subdesenvolupats,  està amenaçant la seguretat alimentària. No obstant, un creixement en l’agricultura generaria un creixement econòmic global i conduiria cap a la reducció de la pobresa. L’objectiu d’aquesta tesis consistia en buscar solucions biològiques sintètiques que podrien ajudar a augmentar els rendiments fotosintètics i l’eficiència en l’ús de l’aigua de les plantes i d’aquesta manera millorar els rendiments dels cultius.

Es va treballar en dues possibles maneres de manipular la planta per tal d’aconseguir un augment en fotosíntesi o eficiència en l’ús de l’aigua (EUA). La primera va ser mitjançant la creació d’un mecanisme de concentració del carboni (MCC) en una planta C3 per tal de reduir l’activitat oxigenasa de la Rubisco i així augmentar la fotosíntesi. La segona va ser explorant les possibilitats de canviar la cinètica de l’estoma per tal de ficar en concordança la conductància estomàtica amb la  demanda de CO2 del mesòfil, així donant la possibilitat d’augmentar tant la fotosíntesis com l’EUA.

Per tal de crear un MCC en una planta C3 es va crear un sistema de transport artificial a la membrana interna del cloroplast. Es va introduir una bomba de Cl-­‐ energitzada per la llum (NpHR) a la membrana interna del cloroplast amb l’objectiu de bombardejar  ions  de  Cl-­‐  a  l’interior  del  cloroplasts.  També  es  va  introduir  un transportador  antiport  que  intercanvia  ions  de  Cl-­‐  per  HCO3-­‐  (AE1)  a  la  mateixa membrana amb l’objectiu d’augmentar els nivells de bicarbonat a l’estroma. Es van generar plantes transgèniques expressant el sistema artificial de membrana descrit però no es va observar un augment de la fotosíntesi en les plantes transformades. Caldria desenvolupar un sistema sintètic per tal de retenir el CO2 al voltant de la Rubisco per a que aquesta el pugui fixar.

Per a canviar la cinètica de l’estoma es va simular, en el model OnGuard, com afectaria canviar les propietats del voltatge dels canals Kin i com afectaria la incorporació de la NpHR al tonoplast i a la membrana plasmàtica de les cèl·∙lules de guarda. En els dos casos es va aconseguir canviar la cinètica de l’estoma. Segons la variació en voltatge de la Kin l’estoma s’obria més ràpidament, però es mantenia obert durant la nit, o bé simplement no s’obria. En canvi, al expressar NpHR a la
 
membrana plasmàtica la velocitat de l’estoma augmentava tan en el tancament com en l’obertura, depenent d’un estímul de llum/foscor, comportament que podria derivar en un augment de la fotosíntesis i l’EUA si fos reproduïble in vivo. Després d’un llarg treball de clonatge es va poder aconseguir crear plantes transgèniques  amb totes les característiques reproduïdes en el model, tant en el cas dels Kin, seleccionant el canal KAT1 com a representant d’aquesta família, com en el cas de la NpHR. Caldria fer un treball exhaustiu d’intercanvi de gasos per comprovar si les plantes generades tenen una millora en l’eficiència fotosintètica i en l’EUA, cosa que no s’ha pogut demostrar encara fins al moment.

# metadata.dc.source[].value
Tesis Doctorals - Departament - Biologia Vegetal

# metadata.dc.title[].value
Synthetic engineering of membrane transport to increase photosynthesis and water use efficiency