Nifedipine Is Involved In The Regulation Of Protein Expression And Cell Wall Construction In Pollen Tubes Of Pinus Bungeana

Pollen tube growth delivers the sperm to the ovule in higher plants which is essential for sexual reproduction. The process is fast and confined to the tip of the tube which made pollen tube to be widely used as a model system in the investigation of polarized tip growth, cell-to-cell communication, intracellular and extracellular signaling transduction. In the past decades, a number of reports have demonstrated that calcium was involved in angiosperm pollen tube growth by mediating the cytoskeleton and endo/exocytosis, while the roles of calcium in gymnosperm pollen growth remain unclear due to the limited evidence thus far, even though they are considered to bear some characteristics ancestral to those of angiosperms. In the present study, by means of cytobiological, proteomics and FTIR analysis, we conducted the investigation on the effect of nifedipine (Nif), a calcium channel blocker, on the Pinus bungeana germination and tube growth. The results showed that Nif with effective dose inhibited P. bungeana pollen germination and tube growth. Normally growing pollen tubes displayed a typical tip-focused cytosolic free Ca²⁺ gradient, while Nif-treated pollen tubes emitted faint fluorescence and the Ca²⁺ gradient dissipated. Additionally, most pollen washed with EGTA before cultured in standard medium germinated normally but the pollen tube growth was significantly inhibited, indicating the inhibition effect of Ca²⁺ store on pollen tube growth was more significant than pollen germination. Calmodulin in the medium could reverse the inhibitory effect from the EGTA. The results indicated the influx of Ca²⁺ and subsequent formation of tip-focused cytosolic free Ca²⁺ gradient were definitely necessary for P. bungeana pollen germination and tube growth. Extracellular calmodulin could promote pollen germination and tube growth. FM4-64 labeling showed bright apical and subapical staining pattern in normal pollen tube rather than V-shaped apical staining reported from the angiosperms. However the FM4-64 staining distributed in the whole tube with Nif-treatment, indicating that Nif might affect the recycle of the endosome. In addition, the FM4-64 labeling process demonstrated that the endocytosis occurred in the tip and sub-tip regions of normal pollen tubes and Nif-treated tubes. TEM observation revealed that the organelles distribution was quite different from angiosperm pollen tube. The preferential occurrence of mitochondria was close to tube wall, the dictyosomes did not show any specific zonation or accumulation in the tube, and the ER distribution was absent of orientation as well. The fusion of paramural bodies with plasma membranes occurred frequently in the tip and sub-tip regions of the control pollen tubes. However, mitochondria and ER in Nif-treated pollen tubes showed disorganization and vacuolation. The affinity of the ribosome for the ER membrane was decreased and the ribosome appeared degranulation and depolymerization. Vacuoles accumulated in the tip region and the cell wall became pronouncedly thin. The fusion rarely occurred in the Nif-treated pollen tubes. Furthermore, pollen tube labeled with phalloidin-TRITC showed that microfilament was coaligned in a longitudeinal array that extended from the amyloplasts in the base of the clear zone, while microfilament in Nif-treated tubes were disrupted in the form of short actin filament. Therefore, it was suggested that the release of Ca2+ was arrested after Nif treatment and then the exocytosis was retarded due to actin microfilaments depolymerization. The immunocytochemical analysis revealed the cell wall of pollen tubes was composed of cellulose, callose, pectin and AGPs. Acidic pectin was distributed in the tube wall except for the apex while esterified pectin was present only in the apex. In Nif-treated tube, acidic pectin appeared in the apex and callose accumulated in the apex as well. FTIR microspectroscopy semi-quantitatively documented that the significant changes in the chemical composition of pollen tube walls occurred in the presence of Nif. There was a marked decrease in polysaccharide content while the content of acid pectin increased slightly, in agreement with the result of immunolabeling with JIM5. SDS-PAGE analysis showed there existed significant difference in the component of cell wall proteins between normal and Nif-treated pollen tubes. Two-dimensional electrophoresis displayed nearly 1000 Coomassie Brilliant Blue-stained protein spots. A total of 50 protein spots were reproducibly differentially displayed after Nif treatment and 28 differentially displayed proteins were identified by mass spectrometry. Among the identified proteins, 12 protein spots were up-regulated upon to Nif treatment while 16 spots were down-regulated. These proteins were grouped into distinct functional categories including proteins involved in the metabolism, cell expansion, stress response, post-translational modification and signaling. The results demonstrated low activity of carbohydrate metabolism andinhibition of ATP production. The proteins VCP, RGP and α-tubulin involved in polysaccharide biosynthesis and vesicle trafficking decreased significantly in Nif-treated tubes, indicating the components participated in exocytosis were affected after the block of calcium channel by Nif. The acidic pectin increased in the apex of Nif-treated tube which reduced the plasticity of the tube wall due to a decreased expression of UDPGDH, an enzyme involved in the synthesis of acidic pectin. We also identified a receptor protein kinase, indicating that Nif may regulate the downstream components by phosphorylation or dephosphorylation to mediate the tube to deliver the sperm to the egg apparatus of the ovule. The results suggested that Nif blocked the calcium channel and inhibit the entry of calcium and cytosolic calcium gradient, which induced the change of protein expression. The exocytosis was arrested by disturbance of the organization of cytoskeleton. As a result, the modifications in the composition of the pollen tube walls were changed and further resulted in the cease of pollen tube growth.