Post-germination Seedling Establishment Research Articles

BBX30/miP1b and BBX31/miP1a form a positive feedback loop with ABI5 to regulate ABA-mediated post-germination seedling growth arrest.

In plants the switch to autotrophic growth involves germination followed by post-germination seedling establishment. When environmental conditions are not favorable, the stress hormone Abscisic Acid (ABA) signals plants to postpone seedling establishment by inducing the expression of the transcription factor ABI5. The levels of ABI5 determine the efficiency of the ABA mediated post-germination developmental growth arrest. The molecular mechanisms regulating the stability and activity of ABI5 during the transition to light are less known. Using genetic, molecular and biochemical approach, we found that two B-box domain containing proteins BBX31 and BBX30 alongwith ABI5 inhibit post-germination seedling establishment in a partially interdependent manner. BBX31 and BBX30 are also characterized as microProteins miP1a and miP1b respectively, based on their small size, single domain and ability to interact with multi-domain proteins. miP1a/BBX31 and miP1b/BBX30 physically interact with ABI5 to stabilize it and promote its binding to promoters of downstream genes. ABI5 reciprocally induces the expression of BBX30 and BBX31 by directly binding to their promoter. ABI5 and the two microProteins thereby form a positive feedback loop to promote ABA-mediated developmental arrest of seedlings.

To grow or not to grow ….

To grow or not to grow ….

CONSTITUTIVELY PHOTOMORPHOGENIC1 promotes ABA-mediated inhibition of post-germination seedling establishment.

Under acute stress conditions, precocious seedling development may result in the premature death of young seedlings, before they switch to autotrophic growth. The phytohormone abscisic acid (ABA) inhibits seed germination and post-germination seedling establishment under unfavorable conditions. Various environmental signals interact with the ABA pathway to optimize these early developmental events under stress. Here, we show that light availability critically influences ABA sensitivity during early seedling development. In dark conditions, the ABA-mediated inhibition of post-germination seedling establishment is strongly enhanced. COP1, a central regulator of seedling development in the dark, is necessary for this enhanced post-germination ABA sensitivity in darkness. Despite their slower germination, cop1 seedlings establish faster than wild type in the presence of ABA in both light and dark. PHY and CRY photoreceptors that inhibit COP1 activity in light modulate ABA-mediated inhibition of seedling establishment in light. Genetically, COP1 acts downstream to ABI5, a key transcriptional regulator of ABA signaling, and does not influence the transcriptional and protein levels of ABI5 during the early post-germination stages. COP1 promotes post-germination growth arrest independent of the antagonistic interaction between ABA and cytokinin signaling pathways. COP1 facilitates the binding of ABI5 on its target promoters and the ABA-mediated upregulation of these target genes is reduced in cop1-4. Together, our results suggest that COP1 positively regulates ABA signaling to inhibit post-germination seedling establishment under stress.

Soybean Mutants Lacking Abundant Seed Storage Proteins Are Impaired in Mobilization of Storage Reserves and Germination.

Spontaneous and radiation-induced mutants of soybean, despite loss of abundant seed proteins, have been reported to grow and reproduce normally without any apparent physiological abnormalities. Here, we report the development and characterization of a soybean line (BSH-2) that lacks several abundant seed storage proteins. One-dimensional and high-resolution two-dimensional gel electrophoresis revealed the absence of the α′ and α subunits of β-conglycinin and G1, G2, G4, and G5 glycinin in the newly developed mutant line (BSH-2). Like our earlier developed soybean mutant line (BSH-3), the seeds of BSH-2 also accumulated high levels of free amino acids as compared with wild-type DN47 seeds. An examination of the germination rates revealed that both BSH-2 and BSH-3 had significantly lower germination rates compared with the parent line DN47. Two-dimensional gel electrophoresis analysis demonstrated that these mutants had slower rates of mobilization of seed storage proteins. The delayed mobilization of storage proteins in BSH-2 and BSH-3 seeds was also correlated with a delayed induction of proteolytic activity in the mutants when compared to DN47. Similarly, qRT-PCR analysis revealed distinct expression pattern of genes involved in proteolytic pathway in the mutants when compared to DN47. Transmission electron microscopy observation of soybean seeds at two germination stages revealed striking differences in the breakdown of protein storage vacuoles and lipid bodies in the mutants. Our study demonstrates that BSH-2 and BSH-3 are compromised in mobilization of storage reserves and the absence of abundant storage proteins may affect the seed germination efficiency and post-germinative seedling establishment.

Peroxisomes in plant reproduction and seed-related development.

Peroxisomes are small multi-functional organelles essential for plant development and growth. Plant peroxisomes play various physiological roles, including phytohormone biosynthesis, lipid catabolism, reactive oxygen species metabolism and many others. Mutant analysis demonstrated key roles for peroxisomes in plant reproduction, seed development and germination and post-germinative seedling establishment; however, the underlying mechanisms remain to be fully elucidated. This review summarizes findings that reveal the importance and complexity of the role of peroxisomes in the pertinent processes. The β-oxidation pathway plays a central role, whereas other peroxisomal pathways are also involved. Understanding the biochemical and molecular mechanisms of these peroxisomal functions will be instrumental to the improvement of crop plants.

ABA Receptor Subfamily III Enhances Abscisic Acid Sensitivity and Improves the Drought Tolerance of Arabidopsis.

The phytohormone abscisic acid (ABA) regulates plant growth, the developmental process, and abiotic stresses. ABA signaling is induced in response to mediate plant acclimation to environmental challenges, including high salinity and drought. The ABA-binding receptors (RCAR/PYR1/PYL), composing of 14 members, are the core components of the ABA-signaling pathway. Here, we observed that the three subfamilies within the RCARs showed different expression patterns at the basal and exogenous ABA levels. Subsequently, we generated transgenic plants overexpressing subfamily III, RCAR11–RCAR14, respectively. The transgenic plants showed increased ABA sensitivity in seed germination and post-germination seedling establishment and root length. Further studies revealed that the overexpressing subfamily III transgenic plants enhanced drought resistance, increased water-use efficiency, and accelerated stress-responsive gene expression compared with the wild-type plants. These findings confirm that the subfamily III plays a key role in ABA-mediated developmental processes and, more importantly, is involved in drought tolerance in the ABA-dependent pathway.

Rodent herbivory differentially affects mortality rates of 14 native plant species with contrasting life history and growth form traits.

Ecosystems are transformed by changes in disturbance regimes including wildfire and herbivory. Rodent consumers can have strong top-down effects on plant community assembly through seed predation, but their impacts on post-germination seedling establishment via seedling herbivory need better characterization, particularly in deserts. To test the legacy effects of fire history, and native rodent consumers on seedling establishment, we evaluated factorial combinations of experimental exclusion of rodents and fire history (burned vs. unburned) on seedling survival of 14 native plant species that vary in their life history strategies and growth form in the Mojave Desert. Seedlings were placed into the experimental plots, and seedling survival was monitored daily for 8days. The legacy effects of fire history had minimal effects on seedling survival, but rodent exclusion, year, and their interaction were strongly significant. Seedling survival rates were nearly sixfold greater in rodent exclusion plots compared to control plots in 2012 (53 vs. 9%) and 17-fold greater in 2013 (17 vs. 1%). The dramatic increase in seedling mortality from 2012 to 2013 was likely driven by an increase in rodent abundance and an outbreak of grasshoppers that appears to have intensified the rodent effect. There was strong variability in plant species survival in response to rodent herbivory with annual plants and forb species showing lower survival than perennial plants and shrub species. These results indicate that rodent consumers can strongly regulate seedling survival of native plant species with potentially strong regulatory effects on plant community development.

The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination.

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 (β-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.

The unique mode of action of a divergent member of the ABA-receptor protein family in ABA and stress signaling

Proteins in the PYR/PYL/RCAR family (PYLs) are known as receptors for the phytohormone ABA. Upon ABA binding, PYL adopts a conformation that allows it to interact with and inhibit clade A protein phosphatase 2Cs (PP2Cs), which are known as the co-receptors for ABA. Inhibition of the PP2Cs then leads to the activation of the SnRK2 family protein kinases that phosphorylate and activate downstream effectors in ABA response pathways. The PYL family has 14 members in Arabidopsis, 13 of which have been demonstrated to function as ABA receptors. The function of PYL13, a divergent member of the family, has been enigmatic. We report here that PYL13 differs from the other PYLs in three key residues that affect ABA perception, and mutations in these three residues can convert PYL13 into a partially functional ABA receptor. Transgenic plants overexpressing PYL13 show increased ABA sensitivity in seed germination and postgermination seedling establishment as well as decreased stomatal conductance, increased water-use efficiency, accelerated stress-responsive gene expression, and enhanced drought resistance. pyl13 mutant plants are less sensitive to ABA inhibition of postgermination seedling establishment. PYL13 interacts with and inhibits some members of clade A PP2Cs (PP2CA in particular) in an ABA-independent manner. PYL13 also interacts with the other PYLs and antagonizes their function as ABA receptors. Our results show that PYL13 is not an ABA receptor but can modulate the ABA pathway by interacting with and inhibiting both the PYL receptors and the PP2C co-receptors.

ATHB17 is a positive regulator of abscisic acid response during early seedling growth.

We performed activation tagging screen to isolate abscisic acid (ABA) response mutants. One of the mutants, designated ahs10 (ABA-hypersensitive 10), exhibited ABA-hypersensitive phenotypes. TAIL-PCR analysis of the mutant revealed that T-DNA was inserted in the promoter region of the Arabidopsis gene, At2g01430, which encodes a homeodomain-leucine zipper protein ATHB17. Subsequent expression analysis indicated that ATHB17 was activated in ahs10. To recapitulate the mutant phenotypes, we prepared ATHB17 OX lines and investigated their phenotypes. The results showed that ATHB17 confers ABA-hypersensitivity and drought tolerance. On the contrary, ATHB17 knockout lines were ABA-insensitive and drought-sensitive, further demonstrating that ATHB17 is involved in ABA and water-stress responses. Interestingly, the ATHB17 effect on seedling growth in the presence of ABA was observed only during the postgermination seedling establishment stage, suggesting that it functions during a narrow developmental window of early seedling growth.

The Plastid Isoform of Triose Phosphate Isomerase Is Required for the Postgerminative Transition from Heterotrophic to Autotrophic Growth in Arabidopsis

During postgerminative seedling establishment, reserves stored during seed filling are mobilized to provide energy and carbon for the growing seedling until autotrophic growth is possible. A plastid isoform of triose phosphate isomerase (pdTPI) plays a crucial role in this transition from heterotrophic to autotrophic growth. A T-DNA insertion in Arabidopsis thaliana pdTPI resulted in a fivefold reduction in transcript, reduced TPI activity, and a severely stunted and chlorotic seedling that accumulated dihydroxyacetone phosphate (DHAP), glycerol, and glycerol-3-phosphate. Methylglyoxal (MG), a by-product of DHAP, also accumulated in the pdtpi mutant. Wild-type seed sown in the presence of any of these four metabolites resulted in a phenocopy of this pdtpi mutant, although MG and DHAP were the most effective based upon dosage. These metabolites (except MG) are by-products of triacylglycerol mobilization and precursors for glycerolipid synthesis, suggesting that lipid metabolism may also be affected. Lipid profiling revealed lower monogalactosyl but higher digalactosyl lipids. It is unclear whether the change in lipid composition is a direct or indirect consequence of the pdtpi mutation, as ribulose-1,5-bis-phosphate carboxylase/oxygenase expression, chloroplast morphology, and starch synthesis are also defective in this mutant. We propose that DHAP and MG accumulation in developing plastids delays the transition from heterotrophic to autotrophic growth, possibly due to MG toxicity.

The regulation of reserve mobilization in cotyledons of lipid-rich Cucumis sativus L. seeds

Conflicting results in the literature regarding the possible regulatory role of the embryonic axis on reserve mobilization in lipid-rich seeds prompted this study on cucumber (Cucumis sativus) seed. Two existing models supporting control by the axis, namely the hormonal control- and source-sink relationship models, were tested by incubating detached cotyledons in plant hormone solutions and axes extracts as well as in the presence of artificial sinks. This approach was an attempt to alleviate any effect that axis removal might have on reserve mobilization in cotyledons measured in terms of changes in fresh and dry weight, respiratory capacity and isocitrate lyase activity. No clear evidence that lipid mobilization in cucumber seeds was controlled by the axis through either a hormonal- or a source-sink mechanism was obtained. However, axis removal prevented the natural shedding of the testa by germinating cucumber seeds resulting in clear differences between intact and detached cotyledons in terms of fresh and dry weight, respiratory and lipid metabolism. Moreover, weakening of the testa on detached cotyledons, by cutting longitudinally, only partially alleviated the inhibitory effect that axis removal had on these metabolic events while total removal alleviated this effect completely. The latter was more pronounced in an elevated oxygen atmosphere indicating that the testa played a more pronounced role in controlling the metabolism of cucumber cotyledons during the post-germinative seedling establishment phase than did the growing axis.

Storage Reserve Mobilisation and Seedling Establishment in Arabidopsis

Storage Reserve Mobilisation and Seedling Establishment in Arabidopsis