Illuminating the cells:transient transformation of citrus to study gene functions and organelle activities related to fruit quality

Although multiple microscopic techniques have been applied to horticultural research,few studies of individual organelles in living fruit cells have been reported to date.In this paper,we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method.Kumquat(Fortunella crassifolia Swingle)was used;it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window.Fruits were transformed with fluorescent reporters,and confocal microscopy and live-cell imaging were used to study their localization and dynamics.Moreover,various pH sensors targeting different subcellular compartments were expressed,and the local pH environments in cells from different plant tissues were compared.The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits.In summary,our method is effective for studying various membrane trafficking events,protein localization,and cell physiology in fruit and can provide new insight into fruit biology research.

IQ67 DOMAIN protein 21 is critical for indentation formation in pavement cell morphogenesis

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule duallocalized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell(PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation width, decreased lobe length, and similar lobe number of PCs, whereas IQD21 overexpression had a different effect on cotyledon PC shape. Weak overexpression led to increased lobe number, decreased indentation width, and similar lobe length, while moderate or great overexpression resulted in decreased lobe number, indentation width, and lobe length of PCs. Live-cell observations revealed that IQD21 accumulation at indentation regions correlates with lobe initiation and outgrowth during PC development. Cell biological and genetic approaches revealed that IQD21 promotes transfacial microtubules anchoring to the plasma membrane via its polybasic sites and bundling at the indentation regions in both periclinal and anticlinal walls. IQD21 controls cortical microtubule organization mainly through promoting Katanin 1-mediated microtubule severing during PC interdigitation. These findings provide the genetic evidence that transfacial microtubule arrays play a determinant role in lobe formation, and the insight into the molecular mechanism of IQD21 in transfacial microtubule organization at indentations and puzzle-shaped PC development.

Profilin Regulates Apical Actin Polymerization to Control Polarized Pollen Tube Growth

Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regu- lation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.

Organizational Innovation of Apical Actin Filaments Drives Rapid Pollen Tube Growth and Turning

Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport during pollen tip growth in Arabidopsis. We found that actin filaments originating from the apical membrane form a specialized structure consisting of longitudinally aligned actin bundles at the cortex and inner cytoplasmic fila- ments with a distinct distribution. Using actin-based pharmacological treatments and genetic mutants in combination with FRAP （fluorescence recovery after photobleaching） technology to visualize the transport of vesicles within the growth domain of pollen tubes, we demonstrated that cortical actin filaments facilitate tip-ward vesicle transport. We also discovered that the inner apical actin filaments prevent backward movement of vesicles, thus ensuring that sufficient vesicles accumulate at the pollen tube tip to support the rapid growth of the pollen tube. The combinatorial effect of cortical and internal apical actin filaments perfectly explains the generation of the inverted ＂V＂ cone-shaped vesicle distribution pattern at the pollen tube tip. When pollen tubes turn, apical actin filaments at the facing side undergo depolymerization and repolymerization to reorient the apical actin structure toward the new growth direction. This actin restructuring precedes vesicle accumulation and changes in tube morphology. Thus, our study provides new insights into the functional relationship between actin dynamics and vesicle transport during rapid and directional pollen tube growth.

Plant villins: Versatile actin regulatory proteins

Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology. Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Villins contain six gelsolin homology domains （G1-G6） and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a defined role in modifying actin dynamics in the pollentube; most of their in vivo activities remain to be ascertained. Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review, we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understand- ing of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.

Calcium imaging in Arabidopsis pollen cells using G-CaMP5

Calcium （Ca2 ＋） signaling has been implicated in poJ]en germination and pollen tube growth. To date, however, we still know very little about how exactly Ca2＋ signaling links to various physiological subcellular processes during pollen germination and pollen tube growth. Given that Ca2＋ signaling is tightly related to the cytosolic concentration and dynamics of Ca2＋, it is vital to trace the dynamic changes in Ca＋ levels in order to decode Ca2＋ signaling. Here, we demonstrate that G-CaMP5 serves well as an indicator for monitoring cytosolic Ca2＋ dynamics in pollen cells. Using this probe, we show that cytosolic Ca2＋ changes dramatically during pollen germination, and, as reported previously, Ca2＋ forms a tip-focused gradient in the pollen tube and undergoes oscillation in the tip region during pollen tube growth. In particular, using G-CaMP5 allowed us to capture the dynamic changes in the cytosolic Ca2＋ concentration （[Ca2＋ ]cyt） in pollen tubes in response to various exogenous treatments. Our data suggest that G-CaMP5 is a suitable probe for monitoring the dynamics of [Ca2＋ ]cyt in pollen cells.