Over-expression of a plant-type phosphoenolpyruvate carboxylase derails Arabidopsis stamen formation.

We examined whether plant-type phosphoenolpyruvate carboxylase (PEPC) is involved in flower organ formation or not by over-expression in Arabidopsis. A wheat PEPC isogene Tappc3A, belonging to the ppc3 group, was targeted due to its preferential expression pattern in pistils and stamens. Transgenic Arabidopsis over-expressing Tappc3A exhibited irregular stamen formation, i.e., a lesser number of stamens per flower and shorter filaments in T2 and T3 generations. Irregular stamens were frequently observed in homozygous T4 lines, but no morphological change was observed in other floral organs. High-degree gene co-expression of Tappc3 isogenes with wheat SEEDSTICKs but not with other homeotic transcription factor genes for flower formation implicates that Tappc3 is under control by the class D genes of the ABCDE model to flower development. In addition, the conservation of CArG box sequences on the Tappc3 promoters supported the developmentally programmed gene expression of ppc3 in wheat flowering organs. Thus, this study provides the first experimental evidence for the critical regulation of plant-type PEPC for flower formation.

Gene co-expression modules behind the three-pistil formation in wheat.

Multi-pistil trait in wheat is of great potential value in plant development research and crop breeding. Our previous studies identified the Pis1 locus that causes three pistils in wheat by genetic mapping using multiple DNA marker systems. However, there are still 26 candidate genes on the locus, and the causal gene remains to be found. In this study, we aimed to approach the molecular mechanism of multi-pistil formation. Comparative RNA sequencing (RNA-Seq) during the pistil formation was undertaken in four wheat lines: a three-pistil mutant TP, a single-pistil TILLING mutant of TP (SP), a three-pistil near-isogenic line CM28TP with the background of cultivar Chunmai 28 (CM28), and CM28. Electron microscopic analysis specified probable developmental stages of young spikes for the three-pistil formation. mRNA sequencing in the young spikes of the four lines represented 253 down-regulated genes and 98 up-regulated genes in both three-pistil lines, which included six potential genes for ovary development. Weighted gene co-expression analysis represented three-pistil trait-associated transcription factor-like genes, among which one hub gene, ARF5, was the most highlighted. ARF5 is on the Pis1 locus and an orthologue of MONOPTEROS which mediates tissue development in Arabidopsis. qRT-PCR validation implies that the deficiency of ARF5 underlies the three-pistil formation in wheat.

The Original Form of C4-Photosynthetic Phosphoenolpyruvate Carboxylase Is Retained in Pooids but Lost in Rice.

Poaceae is the most prominent monocot family that contains the primary cereal crops wheat, rice, and maize. These cereal species exhibit physiological diversity, such as different photosynthetic systems and environmental stress tolerance. Phosphoenolpyruvate carboxylase (PEPC) in Poaceae is encoded by a small multigene family and plays a central role in C4-photosynthesis and dicarboxylic acid metabolism. Here, to better understand the molecular basis of the cereal species diversity, we analyzed the PEPC gene family in wheat together with other grass species. We could designate seven plant-type and one bacterial-type grass PEPC groups, ppc1a, ppc1b, ppc2a, ppc2b, ppc3, ppc4, ppcC4, and ppc-b, respectively, among which ppc1b is an uncharacterized type of PEPC. Evolutionary inference revealed that these PEPCs were derived from five types of ancient PEPCs (ppc1, ppc2, ppc3, ppc4, and ppc-b) in three chromosomal blocks of the ancestral Poaceae genome. C4-photosynthetic PEPC (ppcC4 ) had evolved from ppc1b, which seemed to be arisen by a chromosomal duplication event. We observed that ppc1b was lost in many Oryza species but preserved in Pooideae after natural selection. In silico analysis of cereal RNA-Seq data highlighted the preferential expression of ppc1b in upper ground organs, selective up-regulation of ppc1b under osmotic stress conditions, and nitrogen response of ppc1b. Characterization of wheat ppc1b showed high levels of gene expression in young leaves, transcriptional responses under nitrogen and abiotic stress, and the presence of a Dof1 binding site, similar to ppcC4 in maize. Our results indicate the evolving status of Poaceae PEPCs and suggest the functional association of ppc1-derivatives with adaptation to environmental changes.

Effect of Thermospermine on the Growth and Expression of Polyamine-Related Genes in Rice Seedlings.

A mutant defective in the biosynthesis of thermospermine, acaulis5 (acl5), shows a dwarf phenotype with excess xylem vessels in Arabidopsis thaliana. Exogenous supply of thermospermine remarkably represses xylem differentiation in the root of seedlings, indicating the role of thermospermine in proper repression of xylem differentiation. However, the effect of thermospermine has rarely been investigated in other plant species. In this paper, we examined its effect on the growth and gene expression in rice seedlings. When grown with thermospermine, rice seedlings had no clearly enlarged metaxylem vessels in the root. Expression of OsACL5 was reduced in response to thermospermine, suggesting a negative feedback control of thermospermine biosynthesis like in Arabidopsis. Unlike Arabidopsis, however, rice showed up-regulation of phloem-expressed genes, OsHB5 and OsYSL16, by one-day treatment with thermospermine. Furthermore, expression of OsPAO2 and OsPAO6, encoding extracellular polyamine oxidase whose orthologs are not present in Arabidopsis, was induced by both thermospermine and spermine. These results suggest that thermospermine affects the expression of a subset of genes in rice different from those affected in Arabidopsis.

Polyamine Resistance Is Increased by Mutations in a Nitrate Transporter Gene NRT1.3 (AtNPF6.4) in Arabidopsis thaliana.

Polyamines are small basic compounds present in all living organisms and act in a variety of biological processes. However, the mechanism of polyamine sensing, signaling and response in relation to other metabolic pathways remains to be fully addressed in plant cells. As one approach, we isolated Arabidopsis mutants that show increased resistance to spermine in terms of chlorosis. We show here that two of the mutants have a point mutation in a nitrate transporter gene of the NRT1/PTR family (NPF), NRT1.3 (AtNPF6.4). These mutants also exhibit increased resistance to putrescine and spermidine while loss-of-function mutants of the two closest homologs of NRT1.3, root-specific NRT1.1 (AtNPF6.3) and petiole-specific NRT1.4 (AtNPF6.2), were shown to have a normal sensitivity to polyamines. When the GUS reporter gene was expressed under the control of the NRT1.3 promoter, GUS staining was observed in leaf mesophyll cells and stem cortex cells but not in the epidermis, suggesting that NRT1.3 specifically functions in parenchymal tissues. We further found that the aerial part of the mutant seedling has normal levels of polyamines but shows reduced uptake of norspermidine compared with the wild type. These results suggest that polyamine transport or metabolism is associated with nitrate transport in the parenchymal tissue of the shoot.

Thermospermine modulates expression of auxin-related genes in Arabidopsis.

Thermospermine, a structural isomer of spermine, is widely distributed in the plant kingdom and has been shown to play a role in repressing xylem differentiation by studies of its deficient mutant, acaulis5 (acl5), in Arabidopsis. Our results of microarray and real-time PCR analyses revealed that, in addition to a number of genes involved in xylem differentiation, genes related to auxin signaling were up-regulated in acl5 seedlings. These genes include MONOPTEROS, an auxin response factor gene, which acts as a master switch for auxin-dependent procambium formation, and its target genes. Their expression was reduced by exogenous treatment with thermospermine or by transgenic induction of the ACL5 gene. We examined the effect of synthetic polyamines on the expression of these auxin-related genes and on the vascular phenotype of acl5, and found that tetramines containing the NC3NC3N chain could mimic the effect of thermospermine but longer polyamines containing the same chain had little or no such effect. We also found that thermospermine had an inhibitory effect on lateral root formation in wild-type seedlings and it was mimicked by synthetic tetramines with the NC3NC3N chain. These results suggest the importance of the NC3NC3N chain of thermospermine in its action in modulating auxin signaling.