Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule,nectary, leaf and flower development

The NOOT‐BOP‐COCH‐LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE‐ON‐PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen‐fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference‐mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild‐type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.     

Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus

Functional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain‐of‐function platform, termed ‘iFOX (inducible Full‐length cDNA OvereXpressor gene)‐Hunting’, for inducible expression of B. napus seed cDNAs in Arabidopsis. A Gateway‐compatible plant gene expression vector containing a methoxyfenozide‐inducible constitutive promoter for transgene expression was developed. This vector was used for cloning of random cDNAs from developing B. napus seeds and subsequent Agrobacterium‐mediated transformation of Arabidopsis. The inducible promoter of this vector enabled identification of genes upon induction that are otherwise lethal when constitutively overexpressed and to control developmental timing of transgene expression. Evaluation of a subset of the resulting ~6000 Arabidopsis transformants revealed a high percentage of lines with full‐length B. napus transgene insertions. Upon induction, numerous iFOX lines with visible phenotypes were identified, including one that displayed early leaf senescence. Phenotypic analysis of this line (rsl‐1327) after methoxyfenozide induction indicated high degree of leaf chlorosis. The integrated B. napuscDNA was identified as a homolog of an Arabidopsis acyl‐CoA binding protein (ACBP) gene designated BnACBP1‐like. The early senescence phenotype conferred by BnACBP1‐like was confirmed by constitutive expression of this gene in Arabidopsis and B. napus. Use of the inducible promoter in the iFOX line coupled with RNA‐Seq analyses allowed mechanistic clues and a working model for the phenotype associated with BnACBP1‐like expression. Our results demonstrate the utility of iFOX‐Hunting as a tool for gene discovery and functional characterization of Brassica napus genome.     

Both male and female gametogenesis require a fully functional protein S‐acyl transferase 21 in Arabidopsis thaliana

S‐Acylation is a reversible post‐translational lipid modification in which a long chain fatty acid covalently attaches to specific cysteine(s) of proteins via a thioester bond. It enhances the hydrophobicity of proteins, contributes to their membrane association and plays roles in protein trafficking, stability and signalling. A family of P rotein S‐A cyl T ransferases (PATs) is responsible for this reaction. PATs are multi‐pass transmembrane proteins that possess a catalytic Asp?His?His?Cys cysteine‐rich domain (DHHC‐CRD). In Arabidopsis, there are currently 24 such PATs, five having been characterized, revealing their important roles in growth, development, senescence and stress responses. Here, we report the functional characterization of another PAT, AtPAT21, demonstrating the roles it plays in Arabidopsis sexual reproduction. Loss‐of‐function mutation by T‐DNA insertion in AtPAT21 results in the complete failure of seed production. Detailed studies revealed that the sterility of the mutant is caused by defects in both male and female sporogenesis and gametogenesis. To determine if the sterility observed in atpat21‐1 was caused by upstream defects in meiosis, we assessed meiotic progression in pollen mother cells and found massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. Interestingly, the fragmentation phenotype was substantially reduced in atpat21‐1 spo11‐1 double mutants, indicating that AtPAT21 is required for repair, but not for the formation, of SPO11‐induced meiotic DNA double‐stranded breaks (DSBs) in Arabidopsis. Our data highlight the importance of protein S‐acylation in the early meiotic stages that lead to the development of male and female sporophytic reproductive structures and associated gametophytes in Arabidopsis.     

Transient expression in Nicotiana benthamiana for rapid functional analysis of genes involved in non‐photochemical quenching and carotenoid biosynthesis

Plants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non‐photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor‐intensive and time‐consuming generation of stable transgenic lines and mutant populations – approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ‐related genes from diverse organisms, we performed Agrobacterium tumefaciens‐mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta.     

Artificial selection,pre‐release diet,and gut symbiont inoculation effects on sterile male longevity for area‐wide fruit‐fly management

Longevity is an important life‐history trait for successful and cost‐effective application of the sterile insect technique. Furthermore, it has been shown that females of some species – e.g., Anastrepha ludens (Loew) (Diptera: Tephritidae) – preferentially copulate with ‘old’, sexually experienced males, rather than younger and inexperienced males. Long‐lived sterile males may therefore have greater opportunity to find and mate with wild females than short‐lived males, and be more effective in inducing sterility into wild populations. We explored the feasibility of increasing sterile male lifespan through selection of long‐lived strains and provision of pre‐release diets with added protein, and inoculated with bacterial symbionts recovered from cultures of the gut of wild Anastrepha obliqua (Macquart). Artificial selection for long‐lived A. ludens resulted in a sharp drop of fecundity levels for F1 females. Nevertheless, the cross of long‐lived males with laboratory females produced a female F1 progeny with fecundity levels comparable to those of females in the established colony. However, the male progeny of long‐lived males*laboratory females did not survive in higher proportions than laboratory males. Provision of sugar to A. obliqua adults resulted in increased survival in comparison to adults provided only with water, whereas the addition of protein to sugar‐only diets had no additional effect on longevity. Non‐irradiated males lived longer than irradiated males, and supplying a generic probiotic diet produced no noticeable effect in restoring irradiated male longevity of A. obliqua. We discuss the need to evaluate the time to reach sexual maturity and survival under stress for long‐lived strains, and the inclusion of low amounts of protein and specific beneficial bacteria in pre‐release diets to increase sterile male performance and longevity in the field.     

Size advantage for male function and size‐dependent sex allocation in Ambrosia artemisiifolia,a wind‐pollinated plant

In wind‐pollinated plants, male‐biased sex allocation is often positively associated with plant size and height. However, effects of size (biomass or reproductive investment) and height were not separated in most previous studies. Here, using experimental populations of monoecious plants, Ambrosia altemisiifolia, we examined (1) how male and female reproductive investments (MRI and FRI) change with biomass and height, (2) how MRI and height affect male reproductive success (MRS) and pollen dispersal, and (3) how height affects seed production. Pollen dispersal kernel and selection gradients on MRS were estimated by 2,102 seeds using six microsatellite markers. First, MRI increased with height, but FRI did not, suggesting that sex allocation is more male‐biased with increasing plant height. On the other hand, both MRI and FRI increased with biomass but often more greatly for FRI, and consequently, sex allocation was often female‐biased with biomass. Second, MRS increased with both height and MRI, the latter having the same or larger effect on MRS. Estimated pollen dispersal kernel was fat‐tailed, with the maximum distance between mates tending to increase with MRI but not with height. Third, the number of seeds did not increase with height. Those findings showed that the male‐biased sex allocation in taller plants of A. artemisiifolia is explained by a direct effect of height on MRS.     

Association between putative functional variants in the PSMB9 gene and risk of melanoma – re‐analysis of published melanoma genome‐wide association studies

To mine possibly hidden causal single‐nucleotide polymorphisms (SNPs) of melanoma, we investigated the association of SNPs in 76 M/G1 transition genes with melanoma risk using our published genome‐wide association study (GWAS) data set with 1804 melanoma cases and 1026 cancer‐free controls. We found multiple SNPs with P < 0.01 and performed validation studies for 18 putative functional SNPs in PSMB9 in two other GWAS data sets. Two SNPs (rs1351383 and rs2127675) were associated with melanoma risk in the GenoMEL data set (P = 0.013 and 0.004, respectively), but failed in validation using the Australian data set. Genotype–phenotype analysis revealed these two SNPs were significantly correlated with mRNA expression level of PSMB9. Further experiments revealed that SNP rs2071480, which is in high LD with rs1351383 and rs2127675, may have a weak effect on the promoter activity of PSMB9. Taken together, our data suggested that functional variants in PSMB9 may contribute to melanoma susceptibility.     

ScRALF3, a secreted RALF‐like peptide involved in cell–cell communication between the sporophyte and the female gametophyte in a solanaceous species

Small peptides have been shown to regulate numerous aspects of plant development through cell–cell communication. These signaling events are particularly important during reproduction, regulating gamete development and embryogenesis. Rapid alkalinization factor (RALF)‐like genes, a large gene family that encodes secreted peptides, have specific or ubiquitous expression patterns. Previously, five RALF‐like genes with potential involvement during reproduction were isolated from Solanum chacoense. Here, we show that ScRALF3 is an important peptide regulator of female gametophyte development. Its expression, which is auxin‐inducible, is strictly regulated before and after fertilization. Down‐regulation of ScRALF3 expression by RNA interference leads to the production of smaller fruits that produce fewer seeds, due to improper development of the embryo sacs. Defects include loss of embryo sac nuclei polarization, as well as an increase in asynchronous division, accounting for cellular dysfunctions and premature embryo sac development arrest during megagametogenesis. ScRALF3 is expressed in the sporophytic tissue surrounding the embryo sac, the integument and the nucellus, as revealed by in situ hybridization and GUS staining. As expected for a secreted peptide, fluorescence from an ScRALF3–GFP fusion construct is detected throughout the secretory pathway. Therefore, the ScRALF3 secreted peptide may be directly involved in the regulation of multiple aspects of cell–cell communication between the female gametophyte and its surrounding sporophytic tissue during ovule development.