miR408 is involved in abiotic stress responses in Arabidopsis.

MicroRNAs (miRNAs) are small RNAs that regulate the expression of target genes post-transcriptionally; they are known to play major roles in development and responses to abiotic stress. miR408 is a highly conserved miRNA in plants that responds to the availability of copper and targets genes encoding copper-containing proteins. It was recently recognized to be an important component of the HY5-SPL7 gene network that mediates a coordinated response to light and copper, illustrating its central role in the response of plants to the environment. Expression of miR408 is significantly affected by a variety of developmental and environmental conditions; however, its biological function is unknown. Involvement of miR408 in the abiotic stress response was investigated in Arabidopsis. Expression of miR408, as well as its target genes, was investigated in response to salinity, cold, oxidative stress, drought and osmotic stress. Analyses of transgenic plants with modulated miR408 expression revealed that higher miR408 expression leads to improved tolerance to salinity, cold and oxidative stress, but enhanced sensitivity to drought and osmotic stress. Cellular antioxidant capacity was enhanced in plants with elevated miR408 expression, as manifested by reduced levels of reactive oxygen species and induced expression of genes associated with antioxidative functions, including Cu/Zn superoxide dismutases (CSD1 and CSD2) and glutathione-S-transferase (GST-U25), as well as auxiliary genes: the copper chaperone CCS1 and the redox stress-associated gene SAP12. Overall, the results demonstrate significant involvement of miR408 in abiotic stress responses, emphasizing the central function of miR408 in plant survival.

Differential expression of miRNAs and their target genes in senescing leaves and siliques: insights from deep sequencing of small RNAs and cleaved target RNAs.

MicroRNAs (miRNAs) are a class of small RNAs, which typically function by guiding cleavage of target mRNAs. They are known to play roles in a variety of plant processes including development, responses to environmental stresses and senescence. To identify senescence regulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel analysis of RNA ends libraries, which enable the large-scale examination of miRNA-guided cleavage products, were constructed and sequenced, resulting in over 750 million genome-matched sequences. These large datasets led to the identification a new senescence-inducible small RNA locus, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence.

Programmed cell death occurs asymmetrically during abscission in tomato.

Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.

Localization of the Arabidopsis senescence- and cell death-associated BFN1 nuclease: from the ER to fragmented nuclei.

Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms. However, knowledge of their function is limited. Intracellular localization of the Arabidopsis senescence- and PCD-associated nuclease BFN1 was investigated. Analysis of BFN1-GFP localization in transiently transformed tobacco protoplasts revealed initial localization in filamentous structures spread throughout the cytoplasm, which then clustered around the nuclei as the protoplasts senesced. These filamentous structures were identified as being of ER origin. In BFN1-GFP-transgenic Arabidopsis plants, similar localization of BFN1-GFP was observed in young leaves, that is, in filamentous structures that reorganized around the nuclei only in senescing cells. In late senescence, BFN1-GFP was localized with fragmented nuclei in membrane-wrapped vesicles. BFN1's postulated function as a nucleic acid-degrading enzyme in senescence and PCD is supported by its localization pattern. Our results suggest the existence of a dedicated compartment mediating nucleic acid degradation in senescence and PCD processes.

Identification of defense-related genes newly-associated with tomato flower abscission.

The current abscission model suggests the formation of a post-abscission trans-differentiation of a protective layer as the last step of the process. The present report expands the repertoire of genes activated in the tomato flower abscission zone (AZ), which are likely to be involved in defense responses. We identified four different defense-related genes, including: Cysteine-type endopeptidase, α-Dioxygenase 1 (α-DOX1), HopW-1-1-Interacting protein2 (WIN2), and Stomatal-derived factor-2 (SDF2), that are newly-associated with the late stage of the abscission process. The late expression of these genes, induced at 8-14 h after flower removal when pedicel abscission was already in progress, was AZ-specific, and was inhibited by treatments that prevented pedicel abscission, including 1-methylcyclopropene pretreatment or IAA application. This information supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses.

Microarray analysis of the abscission-related transcriptome in the tomato flower abscission zone in response to auxin depletion.

The abscission process is initiated by changes in the auxin gradient across the abscission zone (AZ) and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the molecular and biochemical basis of the increased AZ sensitivity to ethylene. We examined transcriptome changes in the tomato (Solanum lycopersicum 'Shiran 1335') flower AZ during the rapid acquisition of ethylene sensitivity following flower removal, which depletes the AZ from auxin, with or without preexposure to 1-methylcyclopropene or application of indole-3-acetic acid after flower removal. Microarray analysis using the Affymetrix Tomato GeneChip revealed changes in expression, occurring prior to and during pedicel abscission, of many genes with possible regulatory functions. They included a range of auxin- and ethylene-related transcription factors, other transcription factors and regulatory genes that are transiently induced early, 2 h after flower removal, and a set of novel AZ-specific genes. All gene expressions initiated by flower removal and leading to pedicel abscission were inhibited by indole-3-acetic acid application, while 1-methylcyclopropene pretreatment inhibited only the ethylene-induced expressions, including those induced by wound-associated ethylene signals. These results confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes resulting from auxin depletion. Our results shed light on the regulatory control of abscission at the molecular level and further expand our knowledge of auxin-ethylene cross talk during the initial controlling stages of the process.

Expression analysis of the BFN1 nuclease gene promoter during senescence, abscission, and programmed cell death-related processes.

Little is known about the biological role of nucleases induced during plant senescence and programmed cell death (PCD). Arabidopsis BFN1 has been identified as a senescence-associated type I nuclease, whose protein sequence shares high homology with some other senescence- or PCD-associated plant nucleases. To learn about BFN1 regulation, its expression pattern was analysed. A 2.3 kb portion of the 5' promoter sequence of BFN1 was cloned and its ability to activate the GUS reporter gene was examined. Transgenic Arabidopsis and tomato plants harbouring this chimeric construct were analysed for GUS expression. In both, the BFN1 promoter was able specifically to direct GUS expression in senescent leaves, differentiating xylem and the abscission zone of flowers. Thus, at least part of the regulation of BFN1 is mediated at the transcriptional level, and the regulatory elements are recognized in the two different plants. In tomato, specific expression was observed in the leaf and the fruit abscission zones. The BFN1 promoter was also active in other tissues, including developing anthers and seeds, and in floral organs after fertilization. PCD has been implicated in all of these processes, suggesting that in addition to senescence, BFN1 is involved in PCD associated with different development processes in Arabidopsis.

Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission.

Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.

The characterization of LeNUC1, a nuclease associated with leaf senescence of tomato.

Induction of nuclease and RNase activities, together with decreases in nucleic acid content are considered to be characteristics of senescence in higher plants. However, little is known about the specific identities or functions of the enzymes involved or the mechanisms controlling their activation. Here we report the identification of a 41-kDa-tomato nuclease, LeNUC1, which is specifically induced during tomato leaf senescence but not in ripening fruits. LeNUC1 is a glycoprotein, which can degrade both RNA and DNA and has optimal activity at pH 7.5-8. EDTA inhibits the activity of LeNUC1, while the addition of Co2+ or Mn2+ can restore its activity in the presence of the chelating agent. Interestingly, the activity of LeNUC1 is also induced in young leaves upon treatment with ethylene, which is known to be a senescence-promoting hormone in tomato. Constitutive activity of a 39-kDa nuclease, LeNUC2, similar in its biochemical requirements to LeNUC1, was also detected. LeNUC2 is not induced by ethylene and does not seem to be glycosylated. Based on their characteristics, LeNUC1 and LeNUC2 can be classified as Nuclease I enzymes. LeNUC1 may be involved in nucleic acid metabolism during tomato leaf senescence.