Late spring cold (LSC) is a common climatic phenomenon that refers to the raise of temperature in early spring and the sudden onset of persistent cold temperatures before the start of summer. As Chinese farmers usually carry out large-scale spring sowing in early spring, the start of summer coincides with the sprouting of young crops. The buds of young crops and some fruit trees are not very cold hardy, so the occurrence of LSC usually causes great economic losses to agriculture. Currently, methods to deal with the LSC are usually to build greenhouse or cover with plastic mulch, but these require more labor and are more expensive. Therefore, we wanted to find a way to help farmers cope with the LSC.
Abscisic acid (ABA) is a natural plant hormone and is one of the five natural growth regulators of plants. In plants, ABA plays key roles at low concentration, such as inhibiting plant growth and causing seedlings to go dormant. Some studies have shown that ABA can improve cold and drought tolerance in plants through the RCAR-PP2C pathway. At present, exogenous spraying of ABA is promising in the direction of inhibiting seed germination and promoting fruit production. It can also enhance the cold tolerance of plants when dealing with cold wave, so it is valuable in the prevention of LSC.
Biosynthesis of Abscisic acid
The chemical synthesis of ABA can be produced on a large scale, but due to its special chemical structure, the production process usually produces many toxic wastes and biologically inactive analogs (due to chiral problems). Allowing farmers to directly spray ABA crude products may cause soil contamination that does more harm than good. The difficulty in separating and purifying the crude product results in a high cost of pure ABA, which makes it unsuitable for farmers. Professor Hong Tan's research team at the Chengdu Institute of Biology, Chinese Academy of Sciences (CAS) has realized the biosynthesis of ABA in Botrytis cinerea. During our communication with Prof. Tan, we learned that the biosynthesis of ABA requires complex fermentation steps. Although ABA can be freely transported out of the eukaryotic cell membrane, it is difficult to purify as same as chemical synthesis. At the same time, B. cinerea is a plant pathogenic bacteria, which may lead to widespread plant fungal diseases if biosafety is not met. Prof. Tan also mentioned that the ABA synthesis pathway in B. cinerea has been well studied.
Fig 1. Biosynthesis pathway from FPP to ABA in B. cinerea
Therefore, we thought of a strain commonly used by Chinese farmers for dough fermentation, which is Saccharomyces cerevisiae. As a eukaryotic unicellular organism, it is modified as a chassis organism and also used as a typical eukaryotic cell model in the field of synthetic biology. Thus, we generated the idea of introducing the ABA synthesis pathway from B. cinerea into yeast to construct a strain that could synthesize ABA at the time of dough fermenting. The needed amount of exogenous sprayed ABA is tiny if it is to function as a means of raising the cold tolerance of the plant. Therefore, farmers can use the modified yeast at home to ferment the dough and wash off the ABA with water for spraying, while the fermented dough can also be cooked to kill the yeast.
Eukaryotic exogenous gene polycistronic expression system
Unlike prokaryotes, the natural monocistronic gene expression system of eukaryotes require that an open reading frame (ORF) can express only one gene, and the commonly used strategy is to construct several ORFs one by one into a single plasmid, which is time and labor consumed. In contrast, the polycistronic gene expression system in prokaryotes allows the expression of multiple genes in one ORF. This results in exogenous gene introduction to eukaryotes being much more difficult than prokaryotes. Therefore, we would like to develop a eukaryotic exogenous gene polycistronic expression system. Currently, 2A sequences from virus have already achieved this goal to some extent, but there are some problems such as C-terminal residues and inefficiencies.
Fig 2. The cleavage of Csy4 to CRISPR[1]
RNA-cleavage is wildly used in the construction of cascade RNA and regulation of gene expression in prokaryotic synthetic biology. In these cases, a CRISPR-associated protein, Csy4, show the ability to construct polycistronic expression module and control the expression level of downstream gene accurately. Therefore, we want to give a new application to the CRISPR, i.e., using the high targeting of a 28nt CRISPR by the ssRNA endonuclease Csy4 to achieve the construction of eukaryotic exogenous gene polycistronic expression system. Ultimately, testing the biosynthesis of ABA in yeast using only two ORFs on a single plasmid expressing four enzymes from the BcABA family of B. cinerea.
Through a survey of rural areas in China, we found that farmers do not know the late spring cold (LSC) and the serious agricultural losses it brought. The problem is mainly caused by two reasons: (a) Farmers and local authorities do not receive early warning that LSC is approaching, and they are unable to judge whether LSC will occur from the weather of the previous days. (b)At present, China's meteorological broadcasting systems only report temperature and precipitation, but there is no unified standard and forecast of LSC.
Therefore, SCU-China, based on the LSC prediction model , tries to develop an handy LSC forecast software to help local governments and farmers know the occurrence, intensity and time of LSC in advance so that farmers can prepare for the weather in advance.
Learn more.
1. Qi, Lei et al. “RNA processing enables predictable programming of gene expression.” Nature biotechnology vol. 30,10 (2012): 1002-6. doi:10.1038/nbt.2355
