The Enigmatic Aliphatic Acetogenins and Their Correlations With Lipids During Seed Germination and Leaf Development of Avocado (Persea americana Mill.).

Lipids in avocados have been widely studied due to their nutritional value and several reported bioactivities. Aliphatic acetogenins are a relevant component of the avocado lipidome and have been tested for several potential food and pharma industries applications. This work followed the evolution of avocado fatty acids (FAs) and aliphatic acetogenins during seed germination and leaf growth. Oil extracts of embryonic axes, cotyledons, and leaves from seedlings and trees were divided to analyze free acetylated acetogenins (AcO-acetogenins), and free FAs. Embryonic axes from germinating seeds contained the highest amount of AcO-acetogenins and FAs; this tissue also accumulated the most diverse FA profile with up to 22 detected moieties. Leaves presented the highest variations in AcO-acetogenin profiles during development, although leaves from seedlings accumulated the simplest FA profile with only 10 different FAs. Remarkably, AcO-acetogenins represented half of the carbons allocated to lipids in grown leaves, while embryonic axes and cotyledons always contained more carbons within FAs during germination. Thus, we hypothesized the use of the AcO-acetogenin acyl chain for energy production toward ß-oxidation. Also, α-linolenic and docosahexaenoic acids (DHAs) were proposed as close AcO-acetogenin intermediaries based on a correlation network generated using all these data. Another part of the oil extract was fractionated into different lipid classes before transesterification to profile FAs and acetogenins bound to lipids. Acetogenin backbones were identified for the first time in triglycerides from cotyledons and mainly in polar lipids (which include phospholipids) in all developing avocado tissues analyzed. Seed tissues accumulated preferentially polar lipids during germination, while triglycerides were consumed in cotyledons. Seedling leaves contained minute amounts of triglycerides, and polar lipids increased as they developed. Results from this work suggest acetogenins might be part of the energy and signaling metabolisms, and possibly of membrane structures, underlining the yet to establish role(s) of these unusual lipids in the avocado plant physiology.

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.

Maize E2F transcription factors. Expression, association to promoters of S-phase genes and interaction with the RBR1 protein in chromatin during seed germination.

For seed germination, it is necessary to restart the cell cycle, a process regulated at multiple levels including transcriptional control, that is executed by the E2F family of transcription factors. We identified 12 genes of the E2F family in maize that are expressed differentially during the first 28 h post imbibition (HAI). E2Fa/b1;1 and E2Fc proteins were characterized as an activator and a putative repressor respectively, both forming heterodimers with DPb2 that bind differentially to consensus E2F response elements in promoters of E2F target genes. Transcripts of target genes for these transcription factors accumulate during germination; in dry seeds E2Fc protein is enriched in the target promoters and is replaced by E2Fa/b1;1 as germination advances. RBR1 is found in the same promoters in non-imbibed and 28 HAI seeds, when DNA replication has concluded, and transcription of the E2F targets should stop. During germination promoters of these target genes seem to be decorated with histone marks related to relaxed chromatin structure. Therefore, E2Fs appear to occupy their target genes in a context of open chromatin, with RBR1 fine tuning the progression between the phases.

Optimization of recombinant maize CDKA;1 and CycD6;1 production in Escherichia coli by response surface methodology.

The complex formed by the cyclin-dependent kinase A (CDKA) and cyclin D is responsible for the G1-S transition in the plant cell cycle. Maize (Zea mays L) CDKA; 1 and CycD6; 1 were cloned and expressed in E. coli. The present study describes the optimization of both proteins production using a statistical approach known as response surface methodology (RSM). The experimental design took into account the effects of four variables: optical density of the culture (OD600) before induction, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration, post-induction temperature, and post-induction time. For each protein, a 24 full factorial central composite rotary design for these four independent variables (at five levels each) was employed to fit a polynomial model; which indicated that 30 experiments were required for this procedure. An optimization of CDKA; 1 and CycD6; 1 production levels in the soluble fraction was achieved. Protein conformation and stability were studied by circular dichroism and fluorescence spectroscopy. Finally, in vitro Cyc-CDK complex formation and its kinase activity were confirmed.

Oxidation of proline from the cyclin-binding motif in maize CDKA;1 results in lower affinity with its cyclin regulatory subunit.

Cyclin dependent kinase A; 1 (CDKA; 1) is essential in G1/S transition of cell cycle and its oxidation has been implicated in cell cycle arrest during plant abiotic stress. In the present study, an evaluation at the molecular level was performed to find possible sites of protein oxidative modifications. In vivo studies demonstrated that carbonylation of maize CDKA,1 is associated with a decrease in complex formation with maize cyclin D (CycD). Control and in vitro oxidized recombinant CDKA; 1 were sequenced by mass spectrometry. Proline at the PSTAIRE cyclin-binding motif was identified as the most susceptible oxidation site by comparative analysis of the resulted peptides. The specific interaction between CDKA; 1 and CycD6; 1, measured by surface plasmon resonance (SPR), demonstrated that the affinity and the kinetic of the interaction depended on the reduced-oxidized state of the CDKA; 1. CDKA; 1 protein oxidative modification would be in part responsible for affecting cell cycle progression, and thus producing plant growth inhibition under oxidative stress.

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.

Modulation of CycD3;1-CDK complexes by phytohormones and sucrose during maize germination.

Maize CycD3;1 associates to CDKA or CDKB1;1 proteins during germination and the complexes formed develop kinase activity. These complexes appear to vary in size as germination proceeds, suggesting association to different sets of proteins. CycD3;1 and associated CDK proteins respond to phytohormones and sucrose. Results revealed a reduction in the CycD3;1 protein amount along germination in the presence of indoleacetic acid (IAA) or abscisic acid (ABA), although in the latter protein levels recover at the end of germination. While the levels of CDKA increase with IAA, they decrease with ABA. Both phytohormones, IAA and ABA, increase levels of CDKB1;1 only during the early germination times. CycD3;1 associated kinase activity is only reduced by both phytohormones towards the end of the germination period. On the other hand, lack of sucrose in the imbibition medium strongly reduces CycD3;1 protein levels without affecting the levels of neither CDKA nor CDKB1;1. The corresponding CycD3;1 associated kinase activity is also severely decreased. The presence of sucrose in the medium appears to stabilize the CycD3;1 protein levels.