Overexpressing Vitamin C Defective 2 reduces fertility and alters Ca2+ signals in Arabidopsis pollen.

A potential strategy to mitigate oxidative damage in plants is to increase the abundance of antioxidants, such as ascorbate (i.e. vitamin C). In Arabidopsis (A. thaliana), a rate-limiting step in ascorbate biosynthesis is a phosphorylase encoded by Vitamin C Defective 2 (VTC2). To specifically overexpress VTC2 (VTC2 OE) in pollen, the coding region was expressed using a promoter from a gene with ∼150-fold higher expression in pollen, leading to pollen grains with an eight-fold increased VTC2 mRNA. VTC2 OE resulted in a near-sterile phenotype with a 50-fold decrease in pollen transmission efficiency and a five-fold reduction in the number of seeds per silique. In vitro assays revealed pollen grains were more prone to bursting (greater than two-fold) or produced shorter, morphologically abnormal pollen tubes. The inclusion of a genetically encoded Ca2+ reporter, mCherry-GCaMP6fast (CGf), revealed pollen tubes with altered tip-focused Ca2+ dynamics and increased bursting frequency during periods of oscillatory and arrested growth. Despite these phenotypes, VTC2 OE pollen failed to show expected increases in ascorbate or reductions in reactive oxygen species, as measured using a redox-sensitive dye or a roGFP2. However, mRNA expression analyses revealed greater than two-fold reductions in mRNA encoding two enzymes critical to biosynthetic pathways related to cell walls or glyco-modifications of lipids and proteins: GDP-d-mannose pyrophosphorylase (GMP) and GDP-d-mannose 3',5' epimerase (GME). These results support a model in which the near-sterile defects resulting from VTC2 OE in pollen are associated with feedback mechanisms that can alter one or more signaling or metabolic pathways critical to pollen tube growth and fertility.

Arabidopsis Ca2+-ATPases 1, 2, and 7 in the endoplasmic reticulum contribute to growth and pollen fitness.

Generating cellular Ca2+ signals requires coordinated transport activities from both Ca2+ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca2+-pumps belonging to the Autoinhibited Ca2+-ATPase (ACA) family. Here, we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single-gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, Pathogenesis-related1 (PR1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca2+ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca2+ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca2+ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies.

Nodulin Intrinsic Protein 7;1 Is a Tapetal Boric Acid Channel Involved in Pollen Cell Wall Formation.

Boron is an essential plant micronutrient that plays a structural role in the rhamnogalacturonan II component of the pectic cell wall. To prevent boron deficiency under limiting conditions, its uptake, distribution, and homeostasis are mediated by boric acid transporters and channel proteins. Among the membrane channels that facilitate boric acid uptake are the type II nodulin intrinsic protein (NIP) subfamily of aquaporin-like proteins. Arabidopsis (Arabidopsis thaliana) possesses three NIP II genes (NIP5;1, NIP6;1, and NIP7;1) that show distinct tissue expression profiles (predominantly expressed in roots, stem nodes, and developing flowers, respectively). Orthologs of each are represented in all dicots. Here, we show that purified and reconstituted NIP7;1 is a boric acid facilitator. By using native promoter-reporter fusions, we show that NIP7;1 is expressed predominantly in anthers of young flowers in a narrow developmental window, floral stages 9 and 10, with protein accumulation solely within tapetum cells, where it is localized to the plasma membrane. Under limiting boric acid conditions, loss-of-function T-DNA mutants (nip7;1-1 and nip7;1-2) show reduced fertility, including shorter siliques and an increase in aborted seeds, compared with the wild type. Under these conditions, nip7;1 mutant pollen grains show morphological defects, increased aggregation, defective exine cell wall formation, reduced germination frequency, and decreased viability. During stages 9 and 10, the tapetum is essential for supplying materials to the pollen microspore cell wall. We propose that NIP7;1 serves as a gated boric acid channel in developing anthers that aids in the uptake of this critical micronutrient by tapetal cells.

Arabidopsis thaliana NIP7;1: an anther-specific boric acid transporter of the aquaporin superfamily regulated by an unusual tyrosine in helix 2 of the transport pore.

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9-11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development.

Cloning and characterization of a rice cDNA encoding glutamate decarboxylase.

In this study, we have isolated a rice (Oryza sativa L.) glutamate decarboxylase (RicGAD) clone from a root cDNA library, using a partial Arabidopsis thaliana GAD gene as a probe. The rice root cDNA library was constructed with mRNA, which had been derived from the roots of rice seedlings subjected to phosphorus deprivation. Nucleotide sequence analysis indicated that the RicGAD clone was 1,712 bp long, and harbors a complete open reading frame of 505 amino acids. The 505 amino acid sequence deduced from this RicGAD clone exhibited 67.7 % and 61.9 % identity with OsGAD1 (AB056060) and OsGAD2 (AB056061) in the database, respectively. The 505 amino acid sequence also exhibited 62.9, 64.1, and 64.2 % identity to Arabidopsis GAD (U9937), Nicotiana tabacum GAD (AF020425), and Petunia hybrida GAD (L16797), respectively. The RicGAD was found to possess a highly conserved tryptophan residue, but lacks the lysine cluster at the C-proximal position, as well as other stretches of positively charged residues. The GAD sequence was expressed heterologously using the high copy number plasmid, pVUCH. Our activation analysis revealed that the maximal activation of the RicGAD occurred in the presence of both Ca(2+) and calmodulin. The GAD-encoded 56 approximately 58 kDa protein was identified via Western blot analysis, using an anti-GAD monoclonal antibody. The results of our RT-PCR analyses revealed that RicGAD is expressed predominantly in rice roots obtained from rice seedlings grown under phosphorus deprivation conditions, and in non-germinated brown rice, which is known to have a limited phosphorus bioavailability. These results indicate that RicGAD is a Ca(2+)/ calmodulin-dependent enzyme, and that RicGAD is expressed primarily under phosphate deprivation conditions.

Antimicrobial constituents of Foeniculum vulgare.

A phenyl propanoid derivative, dillapional(1) was found to be a antimicrobial principle of the stems of Foeniculum vulgare (Umbelliferae) with MIC values of 125, 250 and 125/ against Bacillus subtilis, Aspergillus niger and Cladosporium cladosporioides, respectively. A coumarin derivative, scopoletin(2) was also isolated as marginally antimicrobial agent along with inactive compounds, dillapiol(3), bergapten(4), imperatorin(5) and psolaren(6) from this plant. The isolates 1-6 were not active against the Escherichia coli.