Characterization of plasma membrane intrinsic proteins (PIPs) expressed in maize stomata

Aquaporins (AQPs) are small channel proteins that facilitate transmembrane diffusion of H2O and other small solutes, such as H2O2 and CO2. AQPs belonging to the plasma membrane intrinsic protein (PIP) family are subdivided into two groups: PIP1s and PIP2s. When expressed alone in maize, PIP1s are found in the endoplasmic reticulum (ER) whereas PIP2s are localized in the plasma membrane. The co-expression of PIP1s and PIP2s leads to their interaction and their colocalization in the plasma membrane. In maize, RT-qPCR results demonstrated that many PIPs are expressed in maize stomatal complexes and that the expression of PIP1s contributed more than 80% of the total PIP expression. However, the specific expression and function of PIP1s in guard cells (GCs) and subsidiary cells (SCs) are still unknown. In this master thesis, we aim at contributing to the determination of the PIP expression and function in both GCs and SCs. We first verified genetic constructs previously prepared in the lab to generate stable transformed maize lines expressing ZmPIP1;1, ZmPIP1;2, ZmPIP1;3 and ZmPIP1;6 fused to mYFP under control of their respective native promoters. The constructs were tested by biolistic transient expression in maize leaves. We observed mYFP fluorescent signal in epidermal cells and/or subsidiary cells. Further analysis indicated that the signal was probably localized in the ER and around the nucleus. These results indicate that the PIP1 promoters were working. Secondly, we collected separately GCs and SCs by laser microdissection from the maize epidermal strips. Preliminary RT-qPCR results showed that the mRNA levels of ZmPIP1;1, ZmPIP1;2, ZmPIP1;3, ZmPIP1;5, ZmPIP1;6, ZmPIP2;1 and ZmPIP2;2 were higher in SCs than in GCs, suggesting that PIPs play important roles in SCs to regulate stomatal movement. RNAseq data will be obtained to confirm these results. Finally, we generated CRISPR-TSKO genetic constructs to specifically knockout ZmPIP1s in GCs or SCs. Tissue specific promoters and terminators (ZmMUTE for GCs and ZmCST1 for SCs) were amplified from B73 maize genomic DNA and assembled via the Green Gate technology in CRISPR-TSKO to control the expression of Cas9 fused to the mCherry. In addition, four gRNA targeting the six ZmPIP1 genes were cloned. The constructs were tested by transient expression in maize leaves by biolistic. Our results indicated that the SC specific construct was functional as Cas9-mCherry signal was observed in the nucleus. mCherry signal was not observed for the GC specific construct as the ZmMUTE promoter is only active in guard mother cells (at very early stage of stomatal development) and not in mature GCs present in the leaf mature zone that was bombarded in this experiment. Stable transformed maize lines will be generated in the future and stomatal movement will be investigated.