The nucleolar protein SAHY1 is involved in pre-rRNA processing and normal plant growth

Although the nucleolus is involved in ribosome biogenesis, the functions of numerous nucleolus-localized proteins remain unclear. In this study, we genetically isolated Arabidopsis thaliana salt hypersensitive mutant 1 (sahy1), which exhibits slow growth, short roots, pointed leaves, and sterility. SAHY1 encodes an uncharacterized protein that is predominantly expressed in root tips, early developing seeds, and mature pollen grains and is mainly restricted to the nucleolus. Dysfunction of SAHY1 primarily causes the accumulation of 32S, 18S-A₃, and 27SB pre-rRNA intermediates. Coimmunoprecipitation experiments further revealed the interaction of SAHY1 with ribosome proteins and ribosome biogenesis factors. Moreover, sahy1 mutants are less sensitive to protein translation inhibitors and show altered expression of structural constituents of ribosomal genes and ribosome subunit profiles, reflecting the involvement of SAHY1 in ribosome composition and ribosome biogenesis. Analyses of ploidy, S-phase cell cycle progression, and auxin transport and signaling indicated the impairment of mitotic activity, translation of auxin transport carrier proteins, and expression of the auxin-responsive marker DR5::GFP in the root tips or embryos of sahy1 plants. Collectively, these data demonstrate that SAHY1, a nucleolar protein involved in ribosome biogenesis, plays critical roles in normal plant growth in association with auxin transport and signaling.

SALT-HYPERSENSITIVE MUTANT 1, a nucleolar protein involved in ribosome biogenesis, regulates the auxin-mediated development of vegetative and reproductive tissues.     

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.     

Three Classes of Abscisic Acid (ABA)-Insensitive Mutations of Arabidopsis Define Genes that Control Overlapping Subsets of ABA Responses

Wild type and three abscisic acid (ABA)-insensitive mutants of Arabidopsis (ABI1, ABI2, and ABI3) were compared for their ability to respond to ABA for a variety of ABA-inducible responses throughout the life cycle of the plants. The responses tested included effects on seedling growth, proline accumulation in seedlings, ABA-regulated protein synthesis in plantlets, and seed storage protein and lipid synthesis and accumulation. The abi1 and abi2 mutants showed reduced sensitivity to ABA for inhibition of seedling growth, induction of proline accumulation, and alterations in protein synthesis patterns during vegetative growth, but had wild type levels of storage reserves. In contrast, the abi3 mutant had wild type sensitivity for induction of proline accumulation and was only slightly less responsive to ABA with respect to effects on seedling growth and changes in patterns of protein synthesis. The major effects of this mutation were on seed development. Seeds of the abi3 mutant had two-thirds of the wild type level of storage protein and one-third the wild type level of eicosenoic acid, the major fatty acid component of storage lipids in wild type seeds. These results show that none of the abi mutants is insensitive for all ABA-inducible responses and that the abi3 effects are not seed-specific. Comparison of the degree of ABA sensitivity of monogenic mutant lines with that of digenic mutant lines carrying pairwise combinations of the abi mutations suggests that ABA responses in mature seeds are controlled by at least two parallel pathways.     

Mapping and engineering of auxin-induced plasma membrane dissociation in BRX family proteins

Angiosperms have evolved the phloem for the long-distance transport of metabolites. The complex process of phloem development involves genes that only occur in vascular plant lineages. For example, in Arabidopsis thaliana, the BREVIS RADIX (BRX) gene is required for continuous root protophloem differentiation, together with PROTEIN KINASE ASSOCIATED WITH BRX (PAX). BRX and its BRX-LIKE (BRXL) homologs are composed of four highly conserved domains including the signature tandem BRX domains that are separated by variable spacers. Nevertheless, BRX family proteins have functionally diverged. For instance, BRXL2 can only partially replace BRX in the root protophloem. This divergence is reflected in physiologically relevant differences in protein behavior, such as auxin-induced plasma membrane dissociation of BRX, which is not observed for BRXL2. Here we dissected the differential functions of BRX family proteins using a set of amino acid substitutions and domain swaps. Our data suggest that the plasma membrane-associated tandem BRX domains are both necessary and sufficient to convey the biological outputs of BRX function and therefore constitute an important regulatory entity. Moreover, PAX target phosphosites in the linker between the two BRX domains mediate the auxin-induced plasma membrane dissociation. Engineering these sites into BRXL2 renders this modified protein auxin-responsive and thereby increases its biological activity in the root protophloem context.

Phosphosites are responsible for the auxin-induced plasma membrane dissociation observed in the subset of BRX family proteins that function in the differentiation of the root protophloem.     

G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1^KO and ATR^KO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.     

Trigonelline and promotion of cell arrest in G2 of various legumes

Trigonelline, present in dry seeds of Pisum sativum, is transported to enlarging roots and shoots during early seedling ontogeny and promotes cell arrest in G2 in 40% of all root cells. In the absence of trigonelline, this cell population arrests in G1. Results presented herein show that trigonelline also promotes cell arrest in G2 in roots of Glycine max and Phaseolus vulgaris and that the percentage of cells that arrest in G2 in roots of G. max decreases during seedling ontogeny, as it does in P. sativum. During development, trigonelline is synthesized in leaves and is translocated to pods and eventually to seeds during fruit maturation in P. sativum and G. max. Seeds of most legumes have high concentrations of trigonelline and those of some non-legumes have low concentrations.     

Lower Levels of Expression of FATA2 Gene Promote Longer Siliques with Modified Seed Oil Content in Arabidopsis thaliana

Fatty acyl thioesterases control the termination of intraplastidial fatty acid synthesis by hydrolyzing fatty acyl-ACP complexes. The fatty acyl thioesterase A (FATA) gene family in Arabidopsis comprises two members, i.e., FATA1 and FATA2. Previous studies have shown that FATAs display high specificity for unsaturated fatty acids. However, the expression pattern and individual roles of these two FATA genes remains unknown. In this study, we initially studied the expression patterns of FATA1 and FATA2 in various organs of Arabidopsis and we found that FATA1 was expressed at low level in all organs examined and FATA2 was detected in all organs examined, with especially high accumulation in siliques. The transient expression of a FATA2-eGFP fusion in Arabidopsis green leaf protoplasts showed that FATA2 was localized in chloroplasts. A T-DNA insertion mutant line of FATA2 (named fata2) was obtained and used for phenotypic observation. Semiquantitative RT-PCR assay showed that the expression level of FATA2 decreased significantly in fata2 compared with that in wild type. Furthermore, fata2 mutants produced longer siliques with more seeds, whereas seed size was slightly smaller than that of wild type. Compositional analysis of seed oil revealed that, except for a subtly decreased C24:0 and unchanged C22:0 level, all other fatty acids were increased by between 10 and 60 % in fata2 dry seeds compared with those in wild-type. Taken together, our results indicate that FATA2 plays important roles in lipid metabolism in seeds and in silique development in Arabidopsis thaliana.     

CONSTITUTIVE PHOTOMORPHOGENIC 1 promotes seed germination by destabilizing RGA-LIKE 2 in Arabidopsis

Under favorable moisture, temperature, and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGA-LIKE 2 (RGL2), a major repressor of germination in Arabidopsis (Arabidopsis thaliana) seeds. Analysis of seed germination phenotypes of constitutive photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacted directly with RGL2, and in vivo this interaction was strongly enhanced by SUPPRESSOR OF PHYA-105 1. COP1 directly ubiquitinated RGL2 to promote its degradation. Moreover, GA stabilized COP1 with consequent RGL2 destabilization. By uncovering this COP1–RGL2 regulatory module, we reveal a mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

A master regulator of photomorphogenesis positively regulates germination in Arabidopsis seeds by directly ubiquitinating and promoting the degradation of a key repressor of seed germination.     

Lectins and the soybean-Rhizobium symbiosis: I. Immunological investigations of soybean lines,the seeds of which have been reported to lack the 120 000 dalton soybean lectin

Seeds of six soybean lines (Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunofiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lactin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography.Phosphate buffered saline extracts of roots, hypocotyls, stems, and leaves of 3–66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 μg soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were modulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.     

Hormone-Dependent Insertional Mutants of Arabodopsis thalianawith Reduced Viability and Fertility

We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thalianahaving drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant). The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds. The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A. thalianaby exogenous hormones.     

Effect of oligomycin on coupling in isolated mitochondria from oligomycin-resistant mutants of Saccharomyces cerevisiae carrying an oligomycin-resistant ATP phosphohydrolase.

Mutations at either of the two OLI 1 and OLI 2 loci on mitochondrial DNA of Saccharomyces cerevisiae confer high oligomycin resistance to cell growth, but only moderate oligomycin resistance to the ATPase in isolated mitochondria, the degree of resistance being characteristic of each mutant. For the most highly resistant mutant (locus OLI 1) the moderate resistance of the ATPase reaction is considerably magnified at the coupling level in isolated mitochondria. For the mutant at locus OLI 2 the coupling properties exhibit the same oligomycin sensitivity as for the wild type, contrasting with the oligomycin resistance of the ATPase and of cell growth. A conformational hypothesis is proposed to account for this finding.     

MtABCG20 is an ABA exporter influencing root morphology and seed germination of Medicago truncatula

Abscisic acid (ABA) integrates internal and external signals to coordinate plant development, growth and architecture. It plays a central role in stomatal closure, and prevents germination of freshly produced seeds and germination of non‐dormant seeds under unfavorable circumstances. Here, we describe a Medicago truncatula ATP‐binding cassette (ABC) transporter, MtABCG20, as an ABA exporter present in roots and germinating seeds. In seeds, MtABCG20 was found in the hypocotyl–radicle transition zone of the embryonic axis. Seeds of mtabcg20 plants were more sensitive to ABA upon germination, due to the fact that ABA translocation within mtabcg20 embryos was impaired. Additionally, the mtabcg20 produced fewer lateral roots and formed more nodules compared with wild‐type plants in conditions mimicking drought stress. Heterologous expression in Arabidopsis thaliana provided evidence that MtABCG20 is a plasma membrane protein that is likely to form homodimers. Moreover, export of ABA from Nicotiana tabacum BY2 cells expressing MtABCG20 was faster than in the BY2 without MtABCG20. Our results have implications both in legume crop research and determination of the fundamental molecular processes involved in drought response and germination.     

Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants

The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm.