Bioenergetics study of pollen tube growth in Arabidopsis thaliana

Abstract

In higher plants, sexual reproduction requires pollen germination and subsequent pollen tube growth to deliver the sperm cell to the ovule. Pollen tubes have attracted much attention due to their polarized and rapid growth in the last several decades. However, as a non-photosynthetic cell, the pollen tube is not fully understood in terms of its bioenergetics, especially in adenosine triphosphate (ATP) production, nicotinamide adenine dinucleotide phosphate (NADP(H)), and nicotinamide adenine dinucleotide (NAD(H))homeostasis. To study how those energy molecules are coordinated in non-photosynthetic plastids in pollen tubes, here, I introduced the MgATP2- biosensor (At1.03) and modified NADPH (mCherry-iNAP1/4) and NADH/NAD+ (mCherry-SoNar) sensors to different cellular compartments of Arabidopsis pollen tubes. By combining these biosensors and specific inhibitors or mutants, I illustrated how pollen tube plastids obtain ATP, NADH, NADPH, and acetyl-CoA for fatty acid synthesis. Furthermore, I proposed that the pyruvate dehydrogenase (PDH) pathway and mitochondrial respiration are more important in providing acetyl-CoA and ATP to the pollen tube plastid than the PDH bypass and fermentation. Mitochondria are essential organelles for energy supply, and might play an important role in regulating redox homeostasis and NAD(P)H turnover in cells. However, their internal NAD(P)H levels cannot be monitored due to the failure to target biosensors into mitochondria thus far. I, therefore, visualized the endogenous NAD(P)H in the Arabidopsis pollen tube with a multi-photon microscope and found that NAD(P)H is highly correlated with the distribution of mitochondria, both of which peak at the subapex region, and a weak association between NAD(P)H and pollen plastids was also observed. Further studies using Class XI myosin mutants confirmed that altered mitochondrial distribution and trafficking concomitantly affected intracellular NAD(P)H patterns. By targeting the NADPH- and NAD/NAD+-specific biosensors to the pollen tube cytosol of myo11c1-1/myo11c2-1 double mutants, I showed that the growing pollen tubes in the double mutants possessed a lower level of cytosolic NADPH, but a higher cytosolic NADH/NAD+ ratio, than that of the WT. In addition, I observed that the knockout of Myo11C1 and Myo11C2 led to fragmented mitochondria with reduced motility. Therefore, the compromised redox balance could be secondary to the defects in mitochondrial motility, positioning, or morphology. Taken together, these results suggest that the distribution and movement of mitochondria and plastids in Arabidopsis pollen tubes involves Class XI myosins, and their distribution affects the NAD(P)H zoning in growing pollen tubes.