Two cyclin Bs are differentially modulated by glucose and sucrose during maize germination.

Cell proliferation during seed germination is determinant for an appropriate seedling establishment. The present work aimed to evaluate the participation of two maize B-type Cyclins during germination and under the stimulus of two simple sugars: sucrose and glucose. We found out that the corresponding genes, ZmCycB1;2 and ZmCycB2;1, increased their expression at 24 h of germination, but only ZmCycB1;2 responded negatively to sugar type at the highest sugar concentration tested (120 mM). Also, CycB1;2 showed differential protein levels along germination in response to sugar, or its absence. Both CycBs interacted with CDKA;1 and CDKB1;1 by pull down assays. By an immunoprecipitation approach, it was found that each CycB associated with two CDKB isoforms (34 and 36 kDa). A higher proportion of CycB1;2-CDKB-36kDa was coincident to an increased kinase activity in the presence of sugar and particularly in glucose treatment at 36 h of imbibition. CycB1;2-CDKB activity increased in parallel to germination advance and this was dependent on sugar: glucose > sucrose > No sugar treatment. At RAM, CycB1;2 was more abundant in nuclei on Glucose at late germination; DNA-CycB1;2 colocalization was parallel to CycB1;2 inside the nucleus. Overall, results point out CycB1;2 as a player on promoting proliferation during germination by binding a specific CDKB isoform partner and changing its cellular localization to nuclei, co-localizing with DNA, being glucose a triggering signal.

SIPP, a Novel Mitochondrial Phosphate Carrier, Mediates in Self-Incompatibility.

In Solanaceae, the S-specific interaction between the pistil S-RNase and the pollen S-Locus F-box protein controls self-incompatibility (SI). Although this interaction defines the specificity of the pollen rejection response, the identification of three pistil essential modifier genes unlinked to the S-locus (HT-B, 120K, and NaStEP) unveils a higher degree of complexity in the pollen rejection pathway. We showed previously that NaStEP, a stigma protein with homology with Kunitz-type protease inhibitors, is essential to SI in Nicotiana spp. During pollination, NaStEP is taken up by pollen tubes, where potential interactions with pollen tube proteins might underlie its function. Here, we identified NaSIPP, a mitochondrial protein with phosphate transporter activity, as a novel NaStEP-interacting protein. Coexpression of NaStEP and NaSIPP in pollen tubes showed interaction in the mitochondria, although when expressed alone, NaStEP remains mostly cytosolic, implicating NaSIPP-mediated translocation of NaStEP into the organelle. The NaSIPP transcript is detected specifically in mature pollen of Nicotiana spp.; however, in self-compatible plants, this gene has accumulated mutations, so its coding region is unlikely to produce a functional protein. RNA interference suppression of NaSIPP in Nicotiana spp. pollen grains disrupts the SI by preventing pollen tube inhibition. Taken together, our results are consistent with a model whereby the NaStEP and NaSIPP interaction, in incompatible pollen tubes, might destabilize the mitochondria and contribute to arrest pollen tube growth.

Glucose and sucrose differentially modify cell proliferation in maize during germination.

Glucose and sucrose play a dual role: as carbon and energy sources and as signaling molecules. In order to address the impact that sugars may have on maize seeds during germination, embryo axes were incubated with or without either of the two sugars. Expression of key cell cycle markers and protein abundance, cell patterning and de novo DNA synthesis in root meristem zones were analyzed. Embryo axes without added sugars in imbibition medium were unable to grow after 7 days; in sucrose, embryo axes developed seminal and primary roots with numerous root hairs, whereas in glucose axes showed a twisted morphology, no root hair formation but callus-like structures on adventitious and primary seminal roots. More and smaller cells were observed with glucose treatment in root apical meristems. de novo DNA synthesis was stimulated more by glucose than by sucrose. At 24 h of imbibition, expression of ZmCycD2;2a and ZmCycD4;2 was increased by sucrose and reduced by glucose. CDKA1;1 and CDKA2;1 expression was stimulated equally by both sugars. Protein abundance patterns were modified by sugars: ZmCycD2 showed peaks on glucose at 12 and 36 h of imbibition whereas sucrose promoted ZmCycD3 protein accumulation. In presence of glucose ZmCycD3, ZmCycD4 and ZmCycD6 protein abundance was reduced after 24 h. Finally, both sugars stimulated ZmCDKA protein accumulation but at different times. Overall, even though glucose appears to act as a stronger mitogen stimulator, sucrose stimulated the expression of more cell cycle markers during germination. This work provides evidence of a differential response of cell cycle markers to sucrose and glucose during maize germination that may affect the developmental program during plantlet establishment.

A novel motif in the NaTrxh N-terminus promotes its secretion, whereas the C-terminus participates in its interaction with S-RNase in vitro.

BACKGROUND: NaTrxh, a thioredoxin type h, shows differential expression between self-incompatible and self-compatible Nicotiana species. NaTrxh interacts in vitro with S-RNase and co-localizes with it in the extracellular matrix of the stylar transmitting tissue. NaTrxh contains N- and C-terminal extensions, a feature shared by thioredoxin h proteins of subgroup 2. To ascertain the function of these extensions in NaTrxh secretion and protein-protein interaction, we performed a deletion analysis on NaTrxh and fused the resulting variants to GFP. RESULTS: We found an internal domain in the N-terminal extension, called Nß, that is essential for NaTrxh secretion but is not hydrophobic, a canonical feature of a signal peptide. The lack of hydrophobicity as well as the location of the secretion signal within the NaTrxh primary structure, suggest an unorthodox secretion route for NaTrxh. Notably, we found that the fusion protein NaTrxh-GFP(KDEL) is retained in the endoplasmic reticulum and that treatment of NaTrxh-GFP-expressing cells with Brefeldin A leads to its retention in the Golgi, which indicates that NaTrxh uses, to some extent, the endoplasmic reticulum and Golgi apparatus for secretion. Furthermore, we found that Nß contributes to NaTrxh tertiary structure stabilization and that the C-terminus functions in the protein-protein interaction with S-RNase. CONCLUSIONS: The extensions contained in NaTrxh sequence have specific functions on the protein. While the C-terminus directly participates in protein-protein interaction, particularly on its interaction with S-RNase in vitro; the N-terminal extension contains two structurally different motifs: Nα and Nß. Nß, the inner domain (Ala-17 to Pro-27), is essential and enough to target NaTrxh towards the apoplast. Interestingly, when it was fused to GFP, this protein was also found in the cell wall of the onion cells. Although the biochemical features of the N-terminus suggested a non-classical secretion pathway, our results provided evidence that NaTrxh at least uses the endoplasmic reticulum, Golgi apparatus and also vesicles for secretion. Therefore, the Nß domain sequence is suggested to be a novel signal peptide.

The family of maize D-type cyclins: genomic organization, phylogeny and expression patterns.

Cyclin proteins, associated to cyclin-dependent kinases (CDKs), play fundamental roles in cell cycle control as they constitute a very important driving force to allow cell cycle progression. D-type cyclins (CycDs) are important both for interpreting external mitogenic signals and in the control of the G1 phase. The maize (Zea mays) genome appears to contain at least 17 different CycD genes, and they fall into the subgroups previously described for other plants. Maize CycDs have been named according to identity percentages of the corresponding orthologs in rice and Arabidopsis. In silico analysis confirmed the presence of characteristic cyclin domains in each maize CycD gene and showed that their genomic organization is similar to their orthologs in rice and Arabidopsis. The expression of maize CycD genes was followed in seeds, during germination in the presence/absence of exogenously added hormones, and also in different plantlet tissues (mesocotyl, root tips and first leaf). Most cyclins were expressed in germinating seeds and at least in one of the plantlet tissues tested; almost all of the detected cyclins show an accumulating pattern of mRNA along germination (0-24 h) and higher levels in root tissue. Interestingly, some cyclins show high levels in non-proliferating tissues as leaf. Addition of auxins or cytokinins does not seem to importantly modify transcript levels; on the other hand, addition of abscisic acid repressed the expression of several cyclins. The role of each CycD during germination and plant growth and its interaction with other cell cycle proteins becomes a topic of the highest interest.