Makoto Kusaba

Low glutelin content1 : A Dominant Mutation That Suppresses the Glutelin Multigene Family via RNA Silencing in Rice

Low glutelin content1 (Lgc1) is a dominant mutation that reduces glutelin content in rice grains. Glutelin is a major seed storage protein encoded by a multigene family. RNA gel blot and reverse transcriptase–mediated PCR analyses revealed that Lgc1 acts at the mRNA level in a similarity-dependent manner. In Lgc1 homozygotes, there is a 3.5-kb deletion between two highly similar glutelin genes that forms a tail-to-tail inverted repeat, which might produce a double-stranded RNA molecule, a potent inducer of RNA silencing. The hypothesis that Lgc1 suppresses glutelin expression via RNA silencing is supported by transgenic analysis using this Lgc1 candidate region, by reporter gene analysis, and by the detection of small interfering RNAs. In this context, Lgc1 provides an interesting example of RNA silencing occurring among genes that exhibit various levels of similarity to an RNA-silencing–inducing gene. Possible mechanisms for gene silencing of the glutelin multigene family by Lgc1 are discussed.

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway.

Mutants that retain greenness of leaves during senescence are known as “stay-green” mutants. The most famous stay-green mutant is Mendels green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl–protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes.

Genetic analysis of Nicotiana pollen-part mutants is consistent with the presence of an S-ribonuclease inhibitor at the S locus

Self-incompatibility (SI) is a genetic mechanism that restricts inbreeding in flowering plants. In the nightshade family (Solanaceae) SI is controlled by a single multiallelic S locus. Pollen rejection in this system requires the interaction of two S locus products: a stylar (S)-RNase and its pollen counterpart (pollen S). pollen S has not yet been cloned. Our understanding of how this gene functions comes from studies of plants with mutations that affect the pollen but not the stylar SI response (pollen-part mutations). These mutations are frequently associated with duplicated S alleles, but the absence of an obvious additional allele in some plants suggests pollen S can also be deleted. We studied Nicotiana alata plants with an additional S allele and show that duplication causes a pollen-part mutation in several different genetic backgrounds. Inheritance of the duplication was consistent with a competitive interaction model in which any two nonmatching S alleles cause a breakdown of SI when present in the same pollen grain. We also examined plants with presumed deletions of pollen S and found that they instead have duplications that included pollen S but not the S-RNase gene. This finding is consistent with a bipartite structure for the S locus. The absence of pollen S deletions in this study and perhaps other studies suggests that pollen S might be required for pollen viability, possibly because its product acts as an S-RNase inhibitor.

Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis

The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) is the most abundant membrane protein in green plants, and its degradation is a crucial process for the acclimation to high light conditions and for the recovery of nitrogen (N) and carbon (C) during senescence. However, the molecular mechanism of LHCII degradation is largely unknown. Here, we report that chlorophyll b reductase, which catalyzes the first step of chlorophyll b degradation, plays a central role in LHCII degradation. When the genes for chlorophyll b reductases NOL and NYC1 were disrupted in Arabidopsis thaliana, chlorophyll b and LHCII were not degraded during senescence, whereas other pigment complexes completely disappeared. When purified trimeric LHCII was incubated with recombinant chlorophyll b reductase (NOL), expressed in Escherichia coli, the chlorophyll b in LHCII was converted to 7-hydroxymethyl chlorophyll a. Accompanying this conversion, chlorophylls were released from LHCII apoproteins until all the chlorophyll molecules in LHCII dissociated from the complexes. Chlorophyll-depleted LHCII apoproteins did not dissociate into monomeric forms but remained in the trimeric form. Based on these results, we propose the novel hypothesis that chlorophyll b reductase catalyzes the initial step of LHCII degradation, and that trimeric LHCII is a substrate of LHCII degradation.

Transmissible and Nontransmissible Mutations Induced by Irradiating Arabidopsis thaliana Pollen With γ-Rays and Carbon Ions

An early genetic study showed that most radiation-induced mutations are not transmitted to progeny. In recent molecular studies in plants, mainly M2 plants or their progeny, which contain only transmissible mutations, have been analyzed, but the early results imply that these studies are insufficient as comprehensive descriptions of radiation-induced mutations. To study radiation-induced mutations caused by low-LET γ-rays and high-LET carbon ions at the molecular level, we used the pollen-irradiation method and the plant Arabidopsis thaliana to study various mutations, including nontransmissible mutations. This analysis revealed that most mutants induced with irradiation with γ-rays (150–600 Gy) or carbon ions (40–150 Gy) carried extremely large deletions of up to >6 Mbp, the majority of which were not transmitted to progeny. Mutations containing 1- or 4-bp deletions, which were transmitted normally, were also found. Comparison of the deleted regions in the mutants showing various manners of transmission suggests that the nontransmissibility of the large deletions may be due to the deletion of a particular region that contains a gene or genes required for gamete development or viability.

Field transcriptome revealed critical developmental and physiological transitions involved in the expression of growth potential in japonica rice

BackgroundPlant growth depends on synergistic interactions between internal and external signals, and yield potential of crops is a manifestation of how these complex factors interact, particularly at critical stages of development. As an initial step towards developing a systems-level understanding of the biological processes underlying the expression of overall agronomic potential in cereal crops, a high-resolution transcriptome analysis of rice was conducted throughout life cycle of rice grown under natural field conditions.ResultsA wide range of gene expression profiles based on 48 organs and tissues at various developmental stages identified 731 organ/tissue specific genes as well as 215 growth stage-specific expressed genes universally in leaf blade, leaf sheath, and root. Continuous transcriptome profiling of leaf from transplanting until harvesting further elucidated the growth-stage specificity of gene expression and uncovered two major drastic changes in the leaf transcriptional program. The first major change occurred before the panicle differentiation, accompanied by the expression of RFT1, a putative florigen gene in long day conditions, and the downregulation of the precursors of two microRNAs. This transcriptome change was also associated with physiological alterations including phosphate-homeostasis state as evident from the behavior of several key regulators such as miR399. The second major transcriptome change occurred just after flowering, and based on analysis of sterile mutant lines, we further revealed that the formation of strong sink, i.e., a developing grain, is not the major cause but is rather a promoter of this change.ConclusionsOur study provides not only the genetic basis for functional genomics in rice but also new insight into understanding the critical physiological processes involved in flowering and seed development, that could lead to novel strategies for optimizing crop productivity.

A simple and rapid method to detect plant siRNAs using nonradioactive probes

Small interfering RNAs (siRNAs) are key molecules in RNA silencing, which includes posttranscriptional gene silencing, cosuppression, quelling, and RNA interference. The presence of siRNAs indicates RNA silencing in cells. We present a method of detecting siRNAs using nonradioactive probes that involves isolating the small RNA fraction, separating siRNAs using denaturing gel electrophoresis, and performing a Northern blot analysis under low-stringency hybridization conditions. We used digoxigenin-labeled DNA probes for hybridization and detected siRNAs in petunia and rice plants exhibiting silenced phenotypes. This method is a simple and rapid way to detect siRNAs without using radioisotopes.

Stay-green plants: what do they tell us about the molecular mechanism of leaf senescence

A practical approach to increasing crop yields is to extend the duration of active photosynthesis. Stay-green is a term that is used to describe mutant and transgenic plants or cultivars with the trait of maintaining their leaves for a longer period of time than the wild-type or crosses from which they are derived. Analyzing stay-green genotypes contributes to our understanding of the molecular mechanism regulating leaf senescence which may allow us to extend the duration of active photosynthesis in crop plants. This article summarizes recent studies on stay-green plants and the insights they provide on the mechanism of leaf senescence. Briefly, mutations suppressing ethylene, abscisic acid, brassinosteroid, and strigolactone signal transduction or those activating cytokinin signaling often lead to stay-green phenotypes indicating a complex signaling network regulating leaf senescence. Developmentally regulated transcription factors, including NAC or WRKY family members, play key roles in the induction of leaf senescence and thus alteration in the activity of these transcription factors also result in stay-green phenotypes. Impairment in the enzymatic steps responsible for chlorophyll breakdown also leads to stay-green phenotypes. Some of these genotypes die in the middle of the process of chlorophyll breakdown due to the accumulation of toxic intermediates, while others appear to stay-green but their photosynthetic activity declines in a manner similar to wild-type plants. Alterations in certain metabolic pathways in chloroplasts (e.g., photosynthesis) can lead to a delayed onset of leaf senescence with maintenance of photosynthetic activity longer than wild-type plants, indicating that chloroplast metabolism can also affect the regulatory mechanism of leaf senescence.

Characterization of Brassica S‐haplotypes lacking S‐locus glycoprotein1

Self‐incompatibility (SI) in Brassica is regulated by a single multi‐allelic locus, S, which contains highly polymorphic stigma‐expressed genes, SLG and SRK. While SRK is shown to be the determinant of female SI specificity, SLG is thought to assist the function of SRK. Here we report that the SLG genes of self‐incompatible S 18 and S 60 homozygotes of Brassica oleracea have an in‐frame stop codon and a 23 bp deletion resulting in a frame‐shift, respectively. The finding that these SLG genes do not encode functional SLG proteins suggests that SLG is not essential for SI. The possible role of SLG in SI was discussed.

Strigolactone Regulates Leaf Senescence in Concert with Ethylene in Arabidopsis

Prolonged dark treatment induces ethylene synthesis and consequent induction of strigolactone synthesis in the leaf to promote leaf senescence. Leaf senescence is not a passive degenerative process; it represents a process of nutrient relocation, in which materials are salvaged for growth at a later stage or to produce the next generation. Leaf senescence is regulated by various factors, such as darkness, stress, aging, and phytohormones. Strigolactone is a recently identified phytohormone, and it has multiple functions in plant development, including repression of branching. Although strigolactone is implicated in the regulation of leaf senescence, little is known about its molecular mechanism of action. In this study, strigolactone biosynthesis mutant strains of Arabidopsis (Arabidopsis thaliana) showed a delayed senescence phenotype during dark incubation. The strigolactone biosynthesis genes MORE AXIALLY GROWTH3 (MAX3) and MAX4 were drastically induced during dark incubation and treatment with the senescence-promoting phytohormone ethylene, suggesting that strigolactone is synthesized in the leaf during leaf senescence. This hypothesis was confirmed by a grafting experiment using max4 as the stock and Columbia-0 as the scion, in which the leaves from the Columbia-0 scion senesced earlier than max4 stock leaves. Dark incubation induced the synthesis of ethylene independent of strigolactone. Strigolactone biosynthesis mutants showed a delayed senescence phenotype during ethylene treatment in the light. Furthermore, leaf senescence was strongly accelerated by the application of strigolactone in the presence of ethylene and not by strigolactone alone. These observations suggest that strigolactone promotes leaf senescence by enhancing the action of ethylene. Thus, dark-induced senescence is regulated by a two-step mechanism: induction of ethylene synthesis and consequent induction of strigolactone synthesis in the leaf.