NAD⁺ accumulation as a metabolic off switch for orthodox pollen.

Terrestrial plant pollen is classified into two categories based on its metabolic status: pollen with low-metabolism are termed "orthodox" and pollen with high-metabolism are termed "recalcitrant." Nicotinamide adenine dinucleotide (NAD) is crucial for a number of metabolisms in all extant organisms. It has recently been shown that NAD homeostasis plays an important role in a broad range of developmental processes and responses to environment. Recently, a reverse genetic approach shed light on the significance of NAD biosynthesis on pollen fate. In orthodox Arabidopsis pollen, NAD(+) that was accumulated in excess at dispersal dramatically decreased on rehydration. The lack of a key gene that is involved in NAD biosynthesis compromised the excess accumulation. Moreover, absence of the excess accumulation phenocopied the so-called recalcitrant pollen, as demonstrated by the germination inside anthers and the loss of desiccation tolerance. Upon rehydration, NAD(+)-consuming inhibitors impaired tube germination. Taken together, our results suggest that accumulation of NAD(+) functions as a physiochemical molecular switch for suspended metabolism and that the decrease of NAD(+) plays a very important role during transitions in metabolic states. Shifting of the redox state to an oxidizing environment may efficiently control the comprehensive metabolic network underlying the onset of pollen germination.

NAD+ accumulation during pollen maturation in Arabidopsis regulating onset of germination.

Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.

Abolition of the tapetum suicide program ruins microsporogenesis.

Microsporogenesis in angiosperms takes places within the anther. Microspores are surrounded by a layer of cells, the tapetum, which degenerates during the later stages of pollen development with cytological features characteristic of programmed cell death (PCD). We report herein that the expression of AtBI-1, which suppresses Bax-induced cell death, in the tapetum at the tetrad stage inhibits tapetum degeneration and subsequently results in pollen abortion, while activation of AtBI-1 at the later stage does not. Our results demonstrate that the PCD signal commences at the tetrad stage and that the proper timing of PCD in the tapetum is essential for normal microsporogenesis.

Bax-induced cell death of Arabidopsis is meditated through reactive oxygen-dependent and -independent processes.

An Arabidopsis protoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsis mesophyll protoplasts expressing murine Bax cDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivo targeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N -acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of Arabidopsis Bax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.

Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions.

Utilization of transcription factors might be a powerful approach to modification of metabolism for a generation of crops having superior characteristics because a single transcription factor frequently regulates coordinated expression of a set of key genes for respective pathways. Here, we apply the plant-specific Dof1 transcription factor to improve nitrogen assimilation, the essential metabolism including the primary assimilation of ammonia to carbon skeletons to biosynthesize amino acids and other organic compounds involving nitrogen in plants. Expressing Dof1 induced the up-regulation of genes encoding enzymes for carbon skeleton production, a marked increase of amino acid contents, and a reduction of the glucose level in transgenic Arabidopsis. The results suggest cooperative modification of carbon and nitrogen metabolisms on the basis of their intimate link. Furthermore, elementary analysis revealed that the nitrogen content increased in the Dof1 transgenic plants (approximately 30%), indicating promotion of net nitrogen assimilation. Most significantly, the Dof1 transgenic plants exhibit improved growth under low-nitrogen conditions, an agronomically important trait. These results highlight the great utility of transcription factors in engineering metabolism in plants.