NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll – protein complexes during leaf senescence

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll – protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4‐1, a stay‐green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4‐1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation‐related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity‐dependent manner. Surprisingly, the F_v/F_m value remained high in nyc4‐1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4‐1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild‐type and other non‐functional stay‐green mutants, including sgr‐2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll – protein complexes during leaf senescence is discussed.     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Single base substitution in OsCDC48 is responsible for premature senescence and death phenotype in rice

A premature senescence and death 128 (psd128) mutant was isolated from an ethyl methane sulfonate‐induced rice IR64 mutant bank. The premature senescence phenotype appeared at the six‐leaf stage and the plant died at the early heading stage. psd128 exhibited impaired chloroplast development with significantly reduced photosynthetic ability, chlorophyll and carotenoid contents, root vigor, soluble protein content and increased malonaldehyde content. Furthermore, the expression of senescence‐related genes was significantly altered in psd128. The mutant trait was controlled by a single recessive nuclear gene. Using map‐based strategy, the mutation Oryza sativa cell division cycle 48 (OsCDC48) was isolated and predicted to encode a putative AAA‐type ATPase with 809 amino‐acid residuals. A single base substitution at position C2347T in psd128 resulted in a premature stop codon. Functional complementation could rescue the mutant phenotype. In addition, RNA interference resulted in the premature senescence and death phenotype. OsCDC48 was expressed constitutively in the root, stem, leaf and panicle. Subcellular analysis indicated that OsCDC48:YFP fusion proteins were located both in the cytoplasm and nucleus. OsCDC48 was highly conserved with more than 90% identity in the protein levels among plant species. Our results indicated that the impaired function of OsCDC48 was responsible for the premature senescence and death phenotype.     

Identification and molecular mapping of indica high‐tillering dwarf mutant htd4, a mild phenotype allelic mutant of D14 in rice (Oryza sativa L.)

• Metabolism of strigolactones (SLs) can improve the efficiency of nutrient use by regulating the development of roots and shoots in crops, making them an important research focus for molecular breeding. However, as a very important plant hormone, the molecular mechanism of SL signal transduction still remains largely unknown.
• In this study, we isolated an indica high‐tillering dwarf mutant 4 (htd4), a spontaneous mutant of rice, from the restorer line Gui99.
• Mapping and sequencing analysis showed that htd4 was a novel allelic mutant of D14, in which a single base substitution forms a premature termination codon. Quantitative RT‐PCR analyses revealed that expression levels of the genes D10, D17, D27, D3 and D14 increased significantly, while expression of D53 decreased in htd4, compared with the wild type. A subcellular localisation assay showed that the mutant of D14 in htd4 did not disturb the normal localisation of D14 proteins. However, a BiFC assay suggested that the mutant‐type D14 could not interact with D3. Additionally, compared with other D14 allelic mutants, htd4 was the first mutant of D14 discovered in indica, and the differences in many yield traits such as plant height, seed‐setting rate and grain sizes between htd4 and the wild type were less than those between other D14 allelic mutants and the wild type.
• Therefore, htd4 is considered a mild phenotype allelic mutant of D14. We conclude that the absence of functional D14 caused the high‐tillering dwarf phenotype of htd4. Our results may provide vital information for research on D14 function and the application of htd4 in molecular breeding.

     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

Non‐specific phospholipases C,NPC2 and NPC6, are required for root growth in Arabidopsis

Mutants in lipid metabolism often show a lethal phenotype during reproduction that prevents investigating a specific role of the lipid during different developmental processes. We focused on two non‐specific phospholipases C, NPC2 and NPC6, whose double knock‐out causes a gametophyte‐lethal phenotype. To investigate the role of NPC2 and NPC6 during vegetative growth, we produced transgenic knock‐down mutant lines that circumvent the lethal effect during gametogenesis. Despite no defect observed in leaves, root growth was significantly retarded, with abnormal cellular architecture in root columella cells. Furthermore, the short root phenotype was rescued by exogenous supplementation of phosphocholine, a product of non‐specific phospholipase C (NPC) ‐catalyzed phosphatidylcholine hydrolysis. The expression of phospho‐base N‐methyltransferase 1 (PMT1), which produces phosphocholine and is required for root growth, was induced in the knock‐down mutant lines and was attenuated after phosphocholine supplementation. These results suggest that NPC2 and NPC6 may be involved in root growth by producing phosphocholine via metabolic interaction with a PMT‐catalyzed pathway, which highlights a tissue‐specific role of NPC enzymes in vegetative growth beyond the gametophyte‐lethal phenotype.     

Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA‐mediated microtubule depolymerization,stomatal closure,and tolerance response to drought stress

Ubiquitination is a critical post‐translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T‐DNA insertion mutant in the At5g10650 locus. Compared to wild‐type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C‐terminal C3HC4‐type Really Interesting New Gene (RING) motif, which was essential for ABA‐mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule‐associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1‐ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA‐promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild‐type levels of H₂O₂ after ABA treatment. These findings indicated that JUL1 acts downstream of H₂O₂ and calcium in the ABA‐mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H₂O₂, and calcium, which in turn resulted in ABA‐hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild‐type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress.     

The trxG family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine‐tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 (SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late‐flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 (atx1), sdg25 and curly leaf (clf), as well as the demethylase double mutant lsd1‐like1 lsd1‐like2 (ldl1 ldl2). We found that the early‐flowering mutants sdg25, atx1 and clf interact antagonistically with the late‐flowering mutant sdg26, whereas the late‐flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/AGAMOUS‐LIKE 20 (SOC1/AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late‐flowering phenotype.     

OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice

Drought is one of the major abiotic stresses that directly implicate plant growth and crop productivity. Although many genes in response to drought stress have been identified, genetic improvement to drought resistance especially in food crops is showing relatively slow progress worldwide. Here, we reported the isolation of abscisic acid, stress and ripening (ASR) genes from upland rice variety, IRAT109 (Oryza sativa L. ssp. japonica), and demonstrated that overexpression of OsASR5 enhanced osmotic tolerance in Escherichia coli and drought tolerance in Arabidopsis and rice by regulating leaf water status under drought stress conditions. Moreover, overexpression of OsASR5 in rice increased endogenous ABA level and showed hypersensitive to exogenous ABA treatment at both germination and postgermination stages. The production of H₂O₂, a second messenger for the induction of stomatal closure in response to ABA, was activated in overexpression plants under drought stress conditions, consequently, increased stomatal closure and decreased stomatal conductance. In contrast, the loss‐of‐function mutant, osasr5, showed sensitivity to drought stress with lower relative water content under drought stress conditions. Further studies demonstrated that OsASR5 functioned as chaperone‐like protein and interacted with stress‐related HSP40 and 2OG‐Fe (II) oxygenase domain containing proteins in yeast and plants. Taken together, we suggest that OsASR5 plays multiple roles in response to drought stress by regulating ABA biosynthesis, promoting stomatal closure, as well as acting as chaperone‐like protein that possibly prevents drought stress‐related proteins from inactivation.     

A histone methyltransferase inhibits seed germination by increasing PIF1 mRNA expression in imbibed seeds

Phytochrome‐interacting factor 1 (PIF1) inhibits light‐dependent seed germination. The specific function of PIF1 in seed germination is partly due to its high level of expression in imbibed seeds, but the associated regulatory factors have not been identified. Here we show that mutation of the early flowering in short days (EFS) gene, encoding an H3K4 and H3K36 methyltransferase, decreases the level of H3K36me2 and H3K36me3 but not H3K4me3 at the PIF1 locus, reduces the targeting of RNA polymerase II to the PIF1 locus, and reduces mRNA expression of PIF1 in imbibed seeds. Consistently, the efs mutant geminated even under the phyB_off condition, and had an expression profile of PIF1 target genes similar to that of the pif1 mutant. Introduction of an EFS transgene into the efs mutant restored the level of H3K36me2 and H3K36me3 at the PIF1 locus, the high‐level expression of PIF1 mRNA, the expression pattern of PIF1 target genes, and the light‐dependent germination of these seeds. Introduction of a PIF1 transgene into the efs mutant also restored the expression pattern of PIF1 target genes and light‐dependent germination in imbibed seeds, but did not restore the flowering phenotype. Taken together, our results indicate that EFS is necessary for high‐level expression of PIF1 mRNA in imbibed seeds.     

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica,enhances drought tolerance via ABA‐induced stomatal closure by ROS production in Populus

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H₂O₂ production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H₂O₂) production in transgenic poplar plants.     

A detailed analysis of the leaf rolling mutant sll2 reveals complex nature in regulation of bulliform cell development in rice (Oryza sativa L.)

Bulliform cells are large, thin‐walled and highly vacuolated cells, and play an important role in controlling leaf rolling in response to drought and high temperature. However, the molecular mechanisms regulating bulliform cell development have not been well documented. Here, we report isolation and characterisation of a rice leaf‐rolling mutant, named shallot‐like 2 (sll2). The sll2 plants exhibit adaxially rolled leaves, starting from the sixth leaf stage, accompanied by increased photosynthesis and reduced plant height and tiller number. Histological analyses showed shrinkage of bulliform cells, resulting in inward‐curved leaves. The mutant is recessive and revertible at a rate of 9%. The leaf rolling is caused by a T‐DNA insertion. Cloning of the insertion using TAIL‐PCR revealed that the T‐DNA was inserted in the promoter region of LOC_Os07 g38664. Unexpectedly, the enhanced expression of LOC_Os07 g38664 by the 35S enhancer in the T‐DNA is not responsible for the leaf rolling phenotype. Further, the enhancer also exerted a long‐distance effect, including up‐regulation of several bulliform cell‐related genes. sll2 suppressed the outward leaf rolling of oul1 in the sll2oul1 double mutant. We conclude that leaf rolling in sll2 could be a result of the combined effect of multi‐genes, implying a complex network in regulation of bulliform cell development.