VPS36-Dependent Multivesicular Bodies Are Critical for Plasmamembrane Protein Turnover and Vacuolar Biogenesis.

Most eukaryotic cells target ubiquitinated plasma membrane (PM) proteins for vacuolar degradation in response to environmental and developmental cues. This process involves endosomal sorting complexes required for transport (ESCRT). However, little is known about the cellular mechanisms of ESCRTs in plants. Here, we studied the function of one ESCRT-II component, VPS36, which shows ubiquitin-binding activity and may form a putative ESCRT-II with VPS22 and VPS25 in Arabidopsis (Arabidopsis thaliana). Recessive mutation of the ubiquitously expressed VPS36 causes multiple defects, including delayed embryogenesis, defective root elongation, and limited expansion of cotyledons, and these effects can be complemented by its genomic DNA. Abnormal intracellular compartments containing several membrane transporters, including members of the PIN-FORMEDs, AUXIN RESISTANT 1, and PIP1 families, were found in vps36-1 plants. Employing a genetic approach to cross vps36-1/+ with transgenic plants harboring various fluorescent protein-tagged organelle markers, as well as fluorescent probe and ultrastructural approaches, revealed PM proteins in microsomal fractions from vps36-1 seedlings and demonstrated that VPS36 is critical for forming multivesicular bodies and vacuolar biogenesis for protein degradation. Our study shows that functional VPS36 is essential for a proper endosomal sorting pathway and for vacuolar biogenesis in Arabidopsis.

Arabidopsis Qc-SNARE genes BET11 and BET12 are required for fertility and pollen tube elongation.

KEY MESSAGE: BET11 and 12 are required for pollen tube elongation. Pollen tubes are rapidly growing specialized structures that elongate in a polar manner. They play a crucial role in the delivery of sperm cells through the stylar tissues of the flower and into the embryo sac, where the sperm cells are released to fuse with the egg cell and the central cell to give rise to the embryo and the endosperm. Polar growth at the pollen tube tip is believed to result from secretion of materials by membrane trafficking mechanisms. In this study, we report the functional characterization of Arabidopsis BET11 and BET12, two genes that may code for Qc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Double mutants (bet11/bet12) in a homozygous/heterozygous background showed reduced transmission of the mutant alleles, reduced fertilization of seeds, defective embryo development, reduced pollen tube lengths and formation of secondary pollen tubes. Both BET11 and BET12 are required for fertility and development of pollen tubes in Arabidopsis. More experiments are required to dissect the mechanisms involved.

Identification and exploration of pollen tube small proteins encoded by pollination-induced transcripts.

Pollination is composed of cell-cell communication and complicated signaling cascades that regulate pollen tube growth and guidance toward the ovules for double fertilization, and is critical for successful sexual reproduction. Exploring expression profiles of in vivo grown pollen tubes is important. Nevertheless, it is difficult to obtain accessible pollen tubes for profiling studies in most model plants. By taking advantage of the hollow styles of lily (Lilium longiflorum), in vivo pollen tubes harvested from pollinated styles which had been cut open were used here to study their protein and transcript profiles. Pollination quantitatively and qualitatively altered the total protein composition of elongating pollen tubes. cDNAs generated and amplified from total RNAs of 24 h in vivo grown and 12 h in vitro cultured pollen tubes were used for suppression subtractive hybridization analyses and preparation of home-made array chips. Microarray analyses conducted with different probe sets revealed 16 transcripts specifically present and/or enriched in in vivo pollen tubes. Reverse transcription-PCR (RT-PCR), in situ hybridization and Northern blotting were applied to validate their unique pollination-induced expression features. Interestingly, several transcripts were simultaneously detected on the stylar transmitting tract epidermis, where in vivo pollen tubes tightly adhered during pollination. Their deduced amino acid sequences showed that most of them encoded small proteins and could be classified into several families. Transient assay revealed filament-like structures decorated by these proteins and one probably localized in the generative cell. These small peptides might be critical for pollen tube growth during pollination, and further exploration of their biological functions and mechanisms of action are of great interest.

Rice SIZ1, a SUMO E3 ligase, controls spikelet fertility through regulation of anther dehiscence.

• Sumoylation, a post-translational modification, has important functions in both animals and plants. However, the biological function of the SUMO E3 ligase, SIZ1, in rice (Oryza sativa) is still under investigation. • In this study, we employed two different genetic approaches, the use of siz1 T-DNA mutant and SIZ1-RNAi transgenic plants, to characterize the function of rice SIZ1. • Genetic results revealed the co-segregation of single T-DNA insertional recessive mutation with the observed phenotypes in siz1. In addition to showing reduced plant height, tiller number and seed set percentage, both the siz1 mutant and SIZ1-RNAi transgenic plants showed obvious defects in anther dehiscence, but not pollen viability. The anther indehiscence in siz1 was probably a result of defects in endothecium development before anthesis. Interestingly, rice orthologs of AtIRX and ZmMADS2, which are essential for endothecium development during anther dehiscence, were significantly down-regulated in siz1. Compared with the wild-type, the sumoylation profile of high-molecular-weight proteins in mature spikelets was reduced significantly in siz1 and the SIZ1-RNAi line with notably reduced SIZ1 expression. The nuclear localization signal located in the SIZ1 C-terminus was sufficient for its nuclear targeting in bombarded onion epidermis. • The results suggest the functional role of SIZ1, a SUMO E3 ligase, in regulating rice anther dehiscence.

Pollen-specific SKP1-like proteins are components of functional scf complexes and essential for lily pollen tube elongation.

The ubiquitin-proteasome pathway mediates protein degradation and is involved in diverse aspects of plant development and differentiation, including pollen tube elongation and self-incompatibility. We characterized three lily (Lilium longiflorum) SKP1-like genes, LSK1-LSK3, that are specifically expressed in late pollen developmental stages and the elongating pollen tube. The encoded peptide sequences reveal that LSK1-LSK3 share high identity with Arabidopsis ASK1 and contain a putative N-terminal CUL1- and a C-terminal F-box-interacting domain. Yeast two-hybrid and in vitro affinity binding assays revealed that the LSKs associate with lily CULLIN1. In addition, the LSK genes can functionally complement the yeast skp1 deletion mutant YDR328C. To investigate their biological functions in pollen tube elongation, an in vivo approach for green fluorescent protein (GFP)-tagged dominant-negative LSK1-LSK3 was developed. Microprojectile bombardment with N-terminally truncated LSK1-LSK3 (LSK1-LSK3Delta-GFP) significantly retarded pollen tube elongation in both in vitro germination and in vivo self- and cross-pollination after >12 h incubation. Interestingly, elongation of pollen tubes harboring overexpressed LSK2Delta-GFP and LSK3Delta-GFP was substantially inhibited within the self-pollinated styles. The elongation of most LSK2Delta-GFP-transformed pollen tubes could germinate only on the stigmatic surface of self style and showed statistically significant growth arrest as compared with control pollen tubes. Lily exhibits typical gametophytic self-incompatibility via an unknown mechanism, but LSK2 and LSK3 may be involved in this complex machinery. These results suggest critical roles for LSK1-LSK3 in regulating fundamental pollen tube elongation in vitro and in vivo and that the 26S proteasome-mediated protein pathway plays an important role in pollen tube elongation.