ROP (Rho-like GTPases from plants) GTPases are polarly localized key regulators of polar growth in pollen tubes and other cells in plants. However, how ROP GTPases are regulated and how they control polar growth remains to be fully understood. To gain new insights into ROP-dependent mechanisms underlying polar cell growth, we characterized the interactome of ROP1 GTPase that controls Arabidopsis pollen tube (PT) tip growth, an extreme form of polar cell growth. We established an efficient method for culturing Arabidopsis pollen tubes in liquid medium, which was used for immunoprecipitation/mass spectrometry-based identification of ROP1-associated proteins. A total of 654 candidates were isolated from the ROP1 interactome in Arabidopsis pollen tubes, and GO (Gene Ontology) classification and pathway analysis revealed multiple uncharacterized ROP1-dependent processes including translation, cell wall modification, post transcriptional modification, and ion homeostasis, in addition to known ROP1-dependent pathways. The ROP1-interactome data was further supported by the co-expression of the candidate interactors in highly mature pollen with PT germination and growth defects being discovered in 25% (8/32) of the candidate mutant genes. Taken together, our work uncovers valuable information for the identification and functional elucidation of ROP-associated proteins in the regulation of polar growth, and provides a reliable reference to identify critical regulators of polar cell growth in the future.

Pollen dormancy is a vital trait for plant breeding, enabling the use of pollen as a valuable germplasm resource. However, pollen dormancy can be easily disrupted by humidity and the underlying regulatory mechanisms remain poorly understood. Here we report a novel finding that highlights the involvement of ROP1 in the termination of pollen dormancy triggered by humidity. ROP1 overexpression significantly promoted in vitro pollen germination, and shortened pollen dormancy period under high humidity conditions, leading to premature pollen germination within the anthers. Consistently, the knockdown of Rho GAP-REN1 and Rho GDI2 caused premature pollen germination on medium, and the additive effect on pollen germination was further observed when subjected to increased humidity in the ren1 and gdi2 mutants. Conversely, inhibition of ROP1 activity through the expression of Rho GAP-REN1 and Rho GDI2 suppressed in vitro pollen germination. In summary, our work demonstrates that the suppression of ROP1 activity is crucial for maintaining pollen dormancy, will contribute to facilitating the preservation of pollen as a valuable genetic germplasm resource and advancing development of plant breeding.

Gibberellin (GA) biosynthesis and signaling play important roles in seed setting and grain weight; however, how long-distance GA transport contributes to these traits remains poorly understood. Here, we characterized the Nitrate transporter 1/Peptide transporter Family (NPF) protein OsNPF3.5, which mediates GA allocation in rice (Oryza sativa L. var. Nipponbare). OsNPF3.5 was preferentially expressed in the phloem of the leaf blade at the reproductive stage and was responsive to lower temperatures. Ectopic expression of OsNPF3.5 in Xenopus laevis oocytes showed relatively low uptake activity for GA3,4,7 and abscisic acid (ABA) but a significant efflux activity for GA44 across the plasma membrane. Compared with the wild type, pollen fertility, seed-setting rate, 1,000-grain weight, and grain yield were decreased in osnpf3.5. Moreover, functional disruption of OsNPF3.5 essentially decreased GA44 redistribution from the flag leaf blade and was accompanied by decreased levels of GA3 in anthers and GA1 in caryopses. These results suggest that OsNPF3.5 functions as a GA44 efflux transporter promoting GA44 loading into phloem, thus facilitating GA allocation from flag leaf blade to sink organs including anthers and caryopses, which consequently regulates seed setting, 1,000-grain weight, and grain yield. This represents a mechanism by which long-distance GA precursor transport gets involved in rice seed setting and grain weight formation under variable environmental conditions.