Identification of Reference Genes for Precise Expression Analysis during Germination in Chenopodium quinoa Seeds under Salt Stress.

Chenopodium quinoa Willd. (quinoa), a member of the Amaranthaceae family, is an allotetraploid annual plant, endemic to South America. The plant of C. quinoa presents significant ecological plasticity with exceptional adaptability to several environmental stresses, including salinity. The resilience of quinoa to several abiotic stresses, as well as its nutritional attributes, have led to significant shifts in quinoa cultivation worldwide over the past century. This work first defines germination sensu stricto in quinoa where the breakage of the pericarp and the testa is followed by endosperm rupture (ER). Transcriptomic changes in early seed germination stages lead to unstable expression levels in commonly used reference genes that are typically stable in vegetative tissues. Noteworthy, no suitable reference genes have been previously identified specifically for quinoa seed germination under salt stress conditions. This work aims to identify these genes as a prerequisite step for normalizing qPCR data. To this end, germinating seeds from UDEC2 and UDEC4 accessions, with different tolerance to salt, have been analyzed under conditions of absence (0 mM NaCl) and in the presence (250 mM NaCl) of sodium chloride. Based on the relevant literature, six candidate reference genes, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Monensin sensitivity1 (MON1), Polypyrimidine tract-binding protein (PTB), Actin-7 (ACT7), Ubiquitin-conjugating enzyme (UBC), and 18S ribosomal RNA (18S), were selected and assessed for stability using the RefFinder Tool encompassing the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt in the evaluation. The data presented support the suitability of CqACT7 and CqUBC as reference genes for normalizing gene expression during seed germination under salinity stress. These recommended reference genes can be valuable tools for consistent qPCR studies on quinoa seeds.

Mannans and endo-ß-mannanase transcripts are located in different seed compartments during Brassicaceae germination.

MAIN CONCLUSION: Mannans but not endo-ß-mannanases are mainly found in the mucilage layer of two Brassicaceae seeds. Nonetheless, mannanase mobilization from inner to outer seed layers cannot be ruled out. The contribution of endo-ß-mannanase (MAN) genes to the germination of the wild-type Sisymbrium officinale and cultivated Brassica rapa (Brassicaceae) species has been explored. In both species, mannans have been localized to the imbibed external seed coat layer (mucilage) by fluorescence immunolocalization and MAN enzymatic activity increases in seeds as imbibition progresses, reaching a peak before 100% germination is achieved. The MAN gene families have been annotated and the expression of their members analyzed in vegetative and reproductive organs. In S. officinale and B. rapa, MAN2, MAN5, MAN6, and MAN7 transcripts accumulate upon seed imbibition. SoMAN7 is the most expressed MAN gene in S. officinale germinating seeds, as occurs with its ortholog in Arabidopsis thaliana, but in B. rapa, the most abundant transcripts are BrMAN2 and BrMAN5. These genes (MAN2, MAN5, MAN6, and MAN7) are localized, by mRNA in situ hybridization, to the micropylar at the endosperm layer and to the radicle in S. officinale, but in B. rapa, these mRNAs are faintly found to the micropylar living seed coat layer and are mainly present at the radicle tip and the vascular bundles. If the domestication process undergone by B. rapa is responsible for these different MAN expression patterns, upon germination remains to be elucidated. Since mannans and MAN genes are not spatially distributed in the same seed tissues, a movement of MAN enzymes that are synthesized with typical signal peptides from the embryo tissues to the mucilage layer (via apoplastic space) is necessary for the mannans to be hydrolyzed.

A Developmental Switch of Gene Expression in the Barley Seed Mediated by HvVP1 (Viviparous-1) and HvGAMYB Interactions.

The accumulation of storage compounds in the starchy endosperm of developing cereal seeds is highly regulated at the transcriptional level. These compounds, mainly starch and proteins, are hydrolyzed upon germination to allow seedling growth. The transcription factor HvGAMYB is a master activator both in the maturation phase of seed development and upon germination, acting in combination with other transcription factors. However, the precise mechanism controlling the switch from maturation to germination programs remains unclear. We report here the identification and molecular characterization of Hordeum vulgare VIVIPAROUS1 (HvVP1), orthologous to ABA-INSENSITIVE3 from Arabidopsis thaliana HvVP1 transcripts accumulate in the endosperm and the embryo of developing seeds at early stages and in the embryo and aleurone of germinating seeds up to 24 h of imbibition. In transient expression assays, HvVP1 controls the activation of Hor2 and Amy6.4 promoters exerted by HvGAMYB. HvVP1 interacts with HvGAMYB in Saccharomyces cerevisiae and in the plant nuclei, hindering its interaction with other transcription factors involved in seed gene expression programs, like BPBF. Similarly, this interaction leads to a decrease in the DNA binding of HvGAMYB and the Barley Prolamine-Box binding Factor (BPBF) to their target sequences. Our results indicate that the HvVP1 expression pattern controls the full Hor2 expression activated by GAMYB and BPBF in the developing endosperm and the Amy6.4 activation in postgerminative reserve mobilization mediated by GAMYB. All these data demonstrate the participation of HvVP1 in antagonistic gene expression programs and support its central role as a gene expression switch during seed maturation and germination.

The AFL subfamily of B3 transcription factors: evolution and function in angiosperm seeds.

Seed development follows zygotic embryogenesis; during the maturation phase reserves accumulate and desiccation tolerance is acquired. This is tightly regulated at the transcriptional level and the AFL (ABI3/FUS3/LEC2) subfamily of B3 transcription factors (TFs) play a central role. They alter hormone biosynthesis, mainly in regards to abscisic acid and gibberellins, and also regulate the expression of other TFs and/or modulate their downstream activity via protein-protein interactions. This review deals with the origin of AFL TFs, which can be traced back to non-vascular plants such as Physcomitrella patens and achieves foremost expansion in the angiosperms. In green algae, like the unicellular Chlamydomonas reinhardtii or the pluricellular Klebsormidium flaccidum, a single B3 gene and four B3 paralogous genes are annotated, respectively. However, none of them present with the structural features of the AFL subfamily, with the exception of the B3 DNA-binding domain. Phylogenetic analysis groups the AFL TFs into four Major Clusters of Ortologous Genes (MCOGs). The origin and function of these genes is discussed in view of their expression patterns and in the context of major regulatory interactions in seeds of monocotyledonous and dicotyledonous species.

Mitochondrial AtTrxo1 is transcriptionally regulated by AtbZIP9 and AtAZF2 and affects seed germination under saline conditions.

Mitochondrial thioredoxin-o (AtTrxo1) was characterized and its expression examined in different organs of Arabidopsis thaliana. AtTrxo1 transcript levels were particularly high in dry seeds and cotyledons where they reached a maximum 36 h after imbibition with water, coinciding with 50% germination. Expression was lower in seeds germinating in 100 mM NaCl. To gain insight into the transcriptional regulation of the AtTrxo1 gene, a phylogenomic analysis was coupled with the screening of an arrayed library of Arabidopsis transcription factors in yeast. The basic leucine zipper AtbZIP9 and the zinc finger protein AZF2 were identified as putative transcriptional regulators. Transcript regulation of AtbZIP9 and AtAFZ2 during germination was compatible with the proposed role in transcriptional regulation of AtTrxo1. Transient over-expression of AtbZIP9 and AtAZF2 in Nicotiana benthamiana leaves demonstrated an activation effect of AtbZIP9 and a repressor effect of AtAZF2 on AtTrxo1 promoter-driven reporter expression. Although moderate concentrations of salt delayed germination in Arabidopsis wild-type seeds, those of two different AtTrxo1 knock-out mutants germinated faster and accumulated higher H2O2 levels than the wild-type. All these data indicate that AtTrxo1 has a role in redox homeostasis during seed germination under salt conditions.

Arabidopsis DELLA and two HD-ZIP transcription factors regulate GA signaling in the epidermis through the L1 box cis-element.

Gibberellins (GAs) are plant hormones that affect plant growth and regulate gene expression differentially across tissues. To study the molecular mechanisms underlying GA signaling in Arabidopsis thaliana, we focused on a GDSL lipase gene (LIP1) induced by GA and repressed by DELLA proteins. LIP1 contains an L1 box promoter sequence, conserved in the promoters of epidermis-specific genes, that is bound by ATML1, an HD-ZIP transcription factor required for epidermis specification. In this study, we demonstrate that LIP1 is specifically expressed in the epidermis and that its L1 box sequence mediates GA-induced transcription. We show that this sequence is overrepresented in the upstream regulatory regions of GA-induced and DELLA-repressed transcriptomes and that blocking GA signaling in the epidermis represses the expression of L1 box-containing genes and negatively affects seed germination. We show that DELLA proteins interact directly with ATML1 and its paralogue PDF2 and that silencing of both HD-ZIP transcription factors inhibits epidermal gene expression and delays germination. Our results indicate that, upon seed imbibition, increased GA levels reduce DELLA protein abundance and release ATML1/PDF2 to activate L1 box gene expression, thus enhancing germination potential.

Mannans and endo-ß-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination.

Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-ß-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of ß-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24-30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36-42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.

Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase-encoding gene AtMAN7.

Endo-ß-mannanases (MAN; EC. 3.2.1.78) catalyze the cleavage of ß1→4 bonds in mannan polymers and have been associated with the process of weakening the tissues surrounding the embryo during seed germination. In germinating Arabidopsis thaliana seeds, the most highly expressed MAN gene is AtMAN7 and its transcripts are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. Mutants with a T-DNA insertion in AtMAN7 have a slower germination than the wild type. To gain insight into the transcriptional regulation of the AtMAN7 gene, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Brassicaceae MAN7 gene promoters has been done, and these conserved motifs have been used as bait to look for their interacting transcription factors (TFs), using as a prey an arrayed yeast library from A. thaliana. The basic-leucine zipper TF AtbZIP44, but not the closely related AtbZIP11, has thus been identified and its transcriptional activation upon AtMAN7 has been validated at the molecular level. In the knock-out lines of AtbZIP44, not only is the expression of the AtMAN7 gene drastically reduced, but these mutants have a significantly slower germination than the wild type, being affected in the two phases of the germination process, both in the rupture of the seed coat and in the breakage of the micropylar endosperm cell walls. In the over-expression lines the opposite phenotype is observed.

The BdGAMYB protein from Brachypodium distachyon interacts with BdDOF24 and regulates transcription of the BdCathB gene upon seed germination.

MAIN CONCLUSION: BdDOF24 interacting with BdGAMYB regulates the BdCathB gene upon germination. During barley seed germination, hydrolytic enzymes (α-amylases, proteases, etc.) synthesized in the aleurone layer in response to gibberellins (GA), catalyse the mobilization of storage reserves accumulated in the endosperm during seed maturation. In Brachypodium distachyon, the BdCathB gene that encodes a Cathepsin B-like thiol-protease, orthologous to the wheat Al21 and barley HvCathB, is highly induced in germinating seeds and its expression is regulated by transcription factors (TFs) encoded by genes BdGamyb and BdDof24, orthologous to the barley HvGamyb and BPBF-HvDof24, respectively. Transcripts of both TF genes increase during germination and treatments with abscisic acid (ABA) or paclobutrazol (PAC, an inhibitor of GA biosynthesis) decrease mRNA expression of BdGamyb but do not affect that of BdDof24. Besides, proteins BdDOF24 and BdGAMYB interact in yeast-2 hybrid systems and in plant nuclei, and in transient expression assays in aleurone layers BdDOF24 is a transcriptional repressor and BdGAMYB is an activator of the BdCathB promoter, as occurs with the putative orthologous in barley BPBF-HvDOF24 and HvGAMYB. However, when both TFs are co-bombarded, BdDOF24 enhances the activation driven by BdGAMYB while BPBF-HvDOF24 strongly decreases the HvGAMYB-mediated activation of the BdCathB promoter. The different results obtained when BdDOF24 and BPBF-HvDOF24 interact with BdGAMYB and HvGAMYB are discussed.

The AtCathB3 gene, encoding a cathepsin B-like protease, is expressed during germination of Arabidopsis thaliana and transcriptionally repressed by the basic leucine zipper protein GBF1.

Protein hydrolysis plays an important role during seed germination and post-germination seedling establishment. In Arabidopsis thaliana, cathepsin B-like proteases are encoded by a gene family of three members, but only the AtCathB3 gene is highly induced upon seed germination and at the early post-germination stage. Seeds of a homozygous T-DNA insertion mutant in the AtCathB3 gene have, besides a reduced cathepsin B activity, a slower germination than the wild type. To explore the transcriptional regulation of this gene, we used a combined phylogenetic shadowing approach together with a yeast one-hybrid screening of an arrayed library of approximately 1200 transcription factor open reading frames from Arabidopsis thaliana. We identified a conserved CathB3-element in the promoters of orthologous CathB3 genes within the Brassicaceae species analysed, and, as its DNA-interacting protein, the G-Box Binding Factor1 (GBF1). Transient overexpression of GBF1 together with a PAtCathB3::uidA (ß-glucuronidase) construct in tobacco plants revealed a negative effect of GBF1 on expression driven by the AtCathB3 promoter. In stable P35S::GBF1 lines, not only was the expression of the AtCathB3 gene drastically reduced, but a significant slower germination was also observed. In the homozygous knockout mutant for the GBF1 gene, the opposite effect was found. These data indicate that GBF1 is a transcriptional repressor of the AtCathB3 gene and affects the germination kinetics of Arabidopsis thaliana seeds. As AtCathB3 is also expressed during post-germination in the cotyledons, a role for the AtCathB3-like protease in reserve mobilization is also inferred.

Nitrate-induced early transcriptional changes during imbibition in non-after-ripened Sisymbrium officinale seeds.

We have here demonstrated for the first time that nitrate not only accelerates testa rupture of non- AR seeds but also modifies expression pattern of the cell-wall remodeling proteins (mannanases; SoMAN6 and SoMAN7) and key genes belonging to metabolism and signaling of ABA (SoNCED6, SoNCED9, SoCYP707A2 and SoABI5) and GAs (SoGA3ox, SoGA20ox, SoGA2ox and SoRGL2). These results were obtained during Sisymbrium officinale seed imbibition in the absence of endosperm rupture. Exogenous ABA induced a notable inhibition of testa rupture in both absence and presence of nitrate being this effect sharply reversed by GA(4+7). However, nitrate was capable to provoke testa rupture in absence of ABA synthesis. The expression of SoMAN6 and SoMAN7 were positively altered by nitrate. Although ABA synthesis seems apparent at the start of non-AR seed imbibition, taken together the results of SoNCED6, SoNCED9 and SoCYP707A2 expression seem to suggest that nitrate leads to a strong net ABA decrease. Likewise, nitrate positively affected the SoABI5 expression when the SoNCED9 expression was also stimulated. By contrast, at the early and final of imbibition, nitrate clearly inhibited the SoABI5 expression. The expression of SoGA2ox6 and SoGA3ox2 are strongly inhibited by nitrate whereas of SoGA20ox6 was stimulated. On the other hand, SoRGL2 transcript level decreased in the presence of nitrate. Taken together, the results presented here suggest that the nitrate signaling is already operative during the non-AR S. officinale seeds imbibition. The nitrate, in cross-talk with the AR network likely increases the favorable molecular conditions that trigger germination.

Softening-up mannan-rich cell walls.

The softening and degradation of the cell wall (CW), often mannan enriched, is involved in several processes during development of higher plants, such as meristematic growth, fruit ripening, programmed cell death, and endosperm rupture upon germination. Mannans are also the predominant hemicellulosic CW polymers in many genera of green algae. The endosperm CWs of dry seeds often contain mannan polymers, sometimes in the form of galactomannans (Gal-mannans). The endo-ß-mannanases (MANs) that catalyse the random hydrolysis of the ß-linkage in the mannan backbone are one of the main hydrolytic enzymes involved in the loosening and remodelling of CWs. In germinating seeds, the softening of the endosperm seed CWs facilitates the emergence of the elongating radicle. Hydrolysis and mobilization of endosperm Gal-mannans by MANs also provides a source of nutrients for early seedling growth, since Gal-mannan, besides its structural role, serves as a storage polysaccharide. Therefore, the role of mannans and of their hydrolytic enzymes is decisive in the life cycle of seeds. This review updates and discusses the significance of mannans and MANs in seeds and explores the increasing biotechnological potential of MAN enzymes.

A View into Seed Autophagy: From Development to Environmental Responses.

Autophagy is a conserved cellular mechanism involved in the degradation and subsequent recycling of cytoplasmic components. It is also described as a catabolic process implicated in the specific degradation of proteins in response to several stimuli. In eukaryotes, the endoplasmic reticulum accumulates an excess of proteins in response to environmental changes, and is the major cellular organelle at the crossroads of stress responses. Return to proteostasis involves the activation of the Unfolded Protein Response (UPR) and eventually autophagy as a feedback mechanism to relieve protein overaccumulation. Recent publications have focused on the relevance of autophagy in two central processes of seed biology: (i) seed storage protein accumulation upon seed maturation and (ii) reserve mobilization during seed imbibition. Although ER-protein accumulation and the subsequent activation of autophagy resemble the Seed Storage Protein (SSP) deposition during seed maturation, the molecular connection between seed development, autophagy, and seed response to abiotic stresses is still an underexplored field. This mini-review presents current advances in autophagy in seeds, highlighting its participation in the normal course of seed development from embryogenesis to germination. Finally, the function of autophagy in response to the seed environment is also considered, as is its involvement in controlling seed dormancy and germination.

Three endo-ß-mannanase genes expressed in the micropylar endosperm and in the radicle influence germination of Arabidopsis thaliana seeds.

Mannans are hemicellulosic polysaccharides in the plant primary cell wall (CW). Mature seeds, specially their endosperm cells, have CWs rich in mannan-based polymers that confer a strong mechanical resistance for the radicle protrusion upon germination. The rupture of the seed coat and endosperm are two sequential events during the germination of Arabidopsis thaliana. Endo-ß-mannanases (MAN; EC. 3.2.1.78) are hydrolytic enzymes that catalyze cleavage of ß1 â†’ 4 bonds in the mannan-polymer. In the genome of Arabidopsis, the endo-ß-mannanase (MAN) family is represented by eight members. The expression of these eight MAN genes has been systematically explored in different organs of this plant and only four of them (AtMAN7, AtMAN6, AtMAN2 and AtMAN5) are expressed in the germinating seeds. Moreover, in situ hybridization analysis shows that their transcript accumulation is restricted to the micropylar endosperm and to the radicle and this expression disappears soon after radicle emergence. T-DNA insertion mutants in these genes (K.O. MAN7, K.O. MAN6, K.O. MAN5), except that corresponding to AtMAN2 (K.O. MAN2), germinate later than the wild type (Wt). K.O. MAN6 is the most affected in the germination time course with a t (50) almost double than that of the Wt. These data suggest that AtMAN7, AtMAN5 and specially AtMAN6 are important for the germination of A. thaliana seeds by facilitating the hydrolysis of the mannan-rich endosperm cell walls.

Genes involved in ethylene and gibberellins metabolism are required for endosperm-limited germination of Sisymbrium officinale L. seeds: germination in Sisymbrium officinale L. seeds.

The rupture of the seed coat and that of the endosperm were found to be two sequential events in the germination of Sisymbrium officinale L. seeds, and radicle protrusion did not occur exactly in the micropylar area but in the neighboring zone. The germination patterns were similar both in the presence of gibberellins (GA(4+7)) and in presence of ethrel. The analysis of genes involved in GAs synthesis and breakdown demonstrated that (1) SoGA2ox6 expression peaked just prior to radicle protrusion (20-22 h), while SoGA3ox2 and SoGA20ox2 expression was high at early imbibition (6 h) diminishing sharply thereafter; (2) the accumulation of SoGA20ox2 transcript was strongly inhibited by paclobutrazol (PB) as well as by inhibitors of ET synthesis and signaling (IESS) early after imbibition (6 h), while SoGA3ox2 and SoGA2ox6 expression was slowly depressed as germination progressed; (3) ethrel and GA(4+7) positively or negatively affected expression of SoGA3ox2, SoGA20ox2, and SoGA2ox6, depending on the germination period studied. Regarding genes involved in ET synthesis, our results showed that SoACS7 was expressed, just prior to radicle emergence while SoACO2 expression slowly increased as germination progressed. Both genes were strongly inhibited by PB but were almost unaffected by externally added ethrel or GA(4+7). These results suggest that GAs are more important than ET during the early stages of imbibition, while ET is more important at the late phases of germination of S. officinale L. seeds.

Reactive Oxygen Species (ROS) and Nucleic Acid Modifications During Seed Dormancy.

The seed is the propagule of higher plants and allows its dissemination and the survival of the species. Seed dormancy prevents premature germination under favourable conditions. Dormant seeds are only able to germinate in a narrow range of conditions. During after-ripening (AR), a mechanism of dormancy release, seeds gradually lose dormancy through a period of dry storage. This review is mainly focused on how chemical modifications of mRNA and genomic DNA, such as oxidation and methylation, affect gene expression during late stages of seed development, especially during dormancy. The oxidation of specific nucleotides produced by reactive oxygen species (ROS) alters the stability of the seed stored mRNAs, being finally degraded or translated into non-functional proteins. DNA methylation is a well-known epigenetic mechanism of controlling gene expression. In Arabidopsis thaliana, while there is a global increase in CHH-context methylation through embryogenesis, global DNA methylation levels remain stable during seed dormancy, decreasing when germination occurs. The biological significance of nucleic acid oxidation and methylation upon seed development is discussed.

After-ripening alters the gene expression pattern of oxidases involved in the ethylene and gibberellin pathways during early imbibition of Sisymbrium officinale L. seeds.

After-ripening (AR) in Sisymbrium officinale seeds altered SoACS7, SoACO2, SoGA20ox2, SoGA3ox2, and SoGA2ox6 gene expression. Except for SoGA20ox2 expression, which sharply diminished, the expression of the other genes rose during development, particularly that of SoACS7. In contrast, only the SoACO2 and SoGA2ox6 transcripts increased with seed desiccation; the others decreased. AR increased the SoGA3ox2 transcript in dry seed, but dramatically decreased the SoACS7 transcript. At the onset of imbibition, AR inhibited SoACS7 and SoACO2 expression and stimulated that of SoGA20ox2, SoGA3ox2, and SoGA2ox6, demonstrating that the participation of ethylene (ET) and gibberellins (GAs) differs in after-ripened and non-after-ripened seeds. The inhibition of SoACO2 expression in the presence of GA(4+7), paclobutrazol (PB), inhibitors of ET synthesis and signalling (IESS), and notably ET+GA(4+7) indicated ET-GA cross-talk in non-after-ripened seeds. A positive effect of AR in reversing this inhibition was found. The idea of ET-GA cross-talk is also supported by the effect of ET on SoGA3ox2 expression, notably induced by the AR process. In contrast, SoGA20ox2 expression did not appear to be susceptible to AR. SoGA2ox6 expression, poorly known in seeds, suggests that AR prompted an up-regulation under all treatments studied, whereas in non-after-ripened seeds expression was down-regulated. On the other hand, the beta-mannanase (MAN) activity dramatically increased in dry after-ripened seed, being significantly boosted by ET. The absence of MAN inhibition by IESS suggests that although ET seems to be one of the factors controlling MAN, its presence did not appear to be essential. GA(4+7) only increased MAN in seeds which were after-ripened. Here, it is proposed that ET and GAs participate actively in establishing the AR process.

A Possible Role of the Aleurone Expressed Gene HvMAN1 in the Hydrolysis of the Cell Wall Mannans of the Starchy Endosperm in Germinating Hordeum vulgare L. Seeds.

The barley endo-ß-mannanase (MAN) gene family (HvMAN1-6) has been identified and the expression of its members analyzed throughout different plant organs, and upon grain development and germination. The HvMAN1 gene has been found to be highly expressed in developing and germinating grains. The MAN (EC 3.2.1.78) enzymatic activity gets a maximum in grains at 48 h of germination (post-germination event). Immunolocalization of mannan polymers in grains has revealed the presence of these polysaccharides in the endosperm cell walls (CWs). By mRNA in situ hybridization assays, the HvMAN1 transcripts have been localized to the aleurone layer, but not to the dead starchy endosperm cells. These data suggest that MAN1 is synthesized in the aleurone layer during early grain imbibition and moves potentially through the apoplast to the endosperm where the hydrolysis of the mannan polymers takes place after germination sensu stricto. Hence, mannans in the starchy endosperm CWs, besides their structural function, could be used as reserve compounds upon barley post-germination.

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein-protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione.

Functional characterization of FaNIP1;1 gene, a ripening-related and receptacle-specific aquaporin in strawberry fruit.

Strawberry fruit (Fragaria × ananassa) is a soft fruit with high water content at ripe stage (more than 90% of its fresh weight). Aquaporins play an important role in plant water homeostasis, through the facilitation of water transport and solutes. We report the role played by FaNIP1;1 in the receptacle ripening process. The analysis by qRT-PCR of FaNIP1;1 showed that this gene is mainly expressed in fruit receptacle and has a ripening-related expression pattern that was accompanied by an increase in both the abscisic acid and water content of the receptacle throughout fruit ripening. Moreover, FaNIP1;1 was induced in situations of water deficit. Additionally, we show that FaNIP1;1 expression was positively regulated by abscisic acid and negatively regulated by auxins. The water transport capacity of FaNIP1;1 was determined by a stopped-flow spectroscopy in yeast over-expressing FaNIP1;1. Glycerol, H2O2 and boron transport were also demonstrated in yeast. On the other hand, GFP-FaNIP1;1 fusion protein was located in plasma membrane. In conclusion, FaNIP1;1 seems to play an important role increasing the plasma membrane permeability, that allows the water accumulation in the strawberry fruit receptacle throughout the ripening process.