Large-scale identification of leaf senescence-associated genes

Leaf senescence is a form of programmed cell death, and is believed to involve preferential expression of a specific set of "senescence-associated genes" (SAGs). To decipher the molecular mechanisms and the predicted complex network of regulatory pathways involved in the senescence program, we have carried out a large-scale gene identification study in a reference plant, Arabidopsis thaliana. Using suppression subtractive hybridization, we isolated approximately 800 cDNA clones representing SAGs expressed in senescing leaves. Differential expression was confirmed by Northern blot analysis for 130 non-redundant genes. Over 70 of the identified genes have not previously been shown to participate in the senescence process. SAG-encoded proteins are likely to participate in macromolecule degradation, detoxification of oxidative metabolites, induction of defense mechanisms, and signaling and regulatory events. Temporal expression profiles of selected genes displayed several distinct patterns, from expression at a very early stage, to the terminal phase of the senescence syndrome. Expression of some of the novel SAGs, in response to age, leaf detachment, darkness, and ethylene and cytokinin treatment was compared. The large repertoire of SAGs identified here provides global insights about regulatory, biochemical and cellular events occurring during leaf senescence.     

Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflata

Background  

Gene expression in Petunia inflata petals undergoes major changes following compatible pollination. Severe flower wilting occurs reproducibly within 36 hours, providing an excellent model for investigation of petal senescence and programmed cell death. Expression of a number of genes and various enzyme activities involved in the degradation and remobilization of macromolecules have been found to be upregulated during the early stages of petal senescence.     

Integrated Signaling in Flower Senescence: An Overview

Flower senescence is the terminal phase of developmental processes that lead to the death of flower, which include, flower wilting, shedding of flower parts and fading of blossoms. Since it is a rapid process as compared to the senescence of other parts of the plant it therefore provides excellent model system for the study of senescence. During flower senescence, developmental and environmental stimuli enhance the upregulation of catabolic processes causing breakdown and remobilization of cellular constituents. Ethylene is well known to play regulatory role in ethylene-sensitive flowers while in ethylene-insensitive flowers abscisic acid (ABA) is thought to be primary regulator. Subsequent to perception of flower senescence signal, death of petals is accompanied by the loss of membrane permeability, increase in oxidative and decreased level of protective enzymes. The last stages of senescence involve the loss of of nucleic acids (DNA and RNA), proteins and organelles, which is achieved by activation of several nucleases, proteases and wall modifiers. Environmental stimuli such as pollination, drought and other stresses also affect senescence by hormonal imbalance. In this article we have covered the following: perception mechanism and specificity of flower senescence, flower senescence-associated events, like degradation of cell membranes, proteins and nucleic acids, environmental/external factors affecting senescence, like pollination and abiotic stress, hormonal and non-hormonal regulation of flower/petal senescence and finally the senescence associated genes (SAGs) have also been described.Key Words: environmental factors, ethylene, flowers, petals, plant hormones, pollination, programmed cell death, senescence, senescence-associated genes     

Lipid globules on the plastid surface in Iris tepal epidermis cells during tepal maturation and senescence

Epidermis cells in the outer tepals of Iris flowers (Iris × hollandica, cv. Blue Magic) start programmed cell death (PCD) prior to floral opening. The tepals show visible senescence symptoms three days after full opening. Visible senescence coincides with collapse (death) of the upper epidermis cells. In these cells, electron-dense particles (plastoglobuli), membranes, and oil bodies were observed in the plastid interior. Electron-dense globules similar to plastoglobuli, thus apparently mainly consisting of lipids, were found on the plastid surface, from before flower opening until cell death. Such electron-dense globules were also present in the cytosol. The size of some of the globules on the plastid surface increased with time. The globules are likely involved in transfer of lipidic/proteinaceous material from the plastid to the cytosol. As the plastids contained ample oil bodies, up to the time of cell death, cell death was likely not due to lack of reserves. Mitochondrial ultrastructure also remained the same until cell death. The role of mitochondria in PCD is discussed.     

Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues

This study was undertaken to characterize the programmed cell death (PCD) processes that occur during detached and natural on-plant senescence and correlate them with the expression of putative regulatory genes that may be involved in the process. DNA fragmentation and TUNEL analysis of broccoli florets showed that DNA was processed into fragments of approximately 180 bp after 48 h of harvest-induced tissue senescence. Characteristic laddering patterns were also visible in Arabidopsis leaves undergoing natural on-plant senescence and during detached senescence. Several recently isolated plant proteins have been assigned a PCD role, for example, the zinc finger containing protein, LSD1 (lesion simulating disease); Bax inhibitor (BI); and serine palmitoyltransferase (SPT), an enzyme in the sphingolipid signalling pathway. Two cDNAs encoding each of these proteins were isolated from broccoli (BoBI-1, BoBI-2, BoLSD1, BoLSD2, BoSPT1, BoSPT2), and the mRNAs increased during harvest-induced senescence in floret tissue. Expression of the Arabidopsis homologues (AtBI-1, AtLSD1, AtSPT1) were also characterized during detached leaf senescence in Arabidopsis leaves. AtBI-1 expression was constitutively expressed during detached senescence, AtLSD1 expression remained constitutively low, and AtSPT1 expression increased during detached senescence.     

Prohibitin and RACK homologues are up-regulated in trypanosomes induced to undergo apoptosis and in naturally occurring terminally differentiated forms

Two genes have been identified as up-regulated late during ConA-induced apoptosis in procyclic form Trypanosoma brucei rhodesiense. The first represents a homologue of prohibitin, a proto-oncogene originally described in mammals and subsequently in yeast, which is involved in cell-cycle control and senescence. The Trypanosoma prohibitin homologue appears to contain within it a putative death domain. The second gene, homologous to a family of regulatory proteins which are receptors for activated protein kinase C (RACKs), is also shown to be up-regulated in terminally differentiated bloodstream form trypanosomes. These are the first endogenous genes to be identified as up-regulated in programmed cell death (PCD) in unicellular organisms.     

Flower bud opening and senescence in roses (Rosa hybrida L.)

The flower is the most significant and beautiful part of plants. Flowers are very useful organs in plant developmental phenomenon. During flower bud opening, various events takes place in a well defined sequence, representing all aspects of plant development, such as cell division, cellular differentiation, cell elongation or expansion and a wide spectrum of gene expression. The complexity of flower bud opening illustrates that various biological mechanisms are involved at different stages. Senescence represents the ultimate stage of floral development and results in wilting or abscission of whole flower or flower parts. Senescence is an active process and governed by a well defined cell death program. Once a flower bud opens, the programmed senescence of petal allows the removal of a metabolically active tissue. In leaves, this process can be reversed, but in floral tissue it cannot, indicating that a highly controlled genetic program for cell death is operating. The termination of a flower involves at least two, sometimes overlapping, mechanisms. In one, the perianth abscises before the majority of its cells initiate a cell death program. Abscission may occur before or during the mobilization of food reserves to other parts of the plant. Alternatively, the petals may be more persistent, so that cell deterioration and food remobilization occur while the petals are still part of the flower. The overall pattern of floral opening varies widely between plant genera, therefore, a number of senescence parameters have been used to group plants into somewhat arbitrary categories. Opening and senescence of rose flower is still an unsolved jigsaw in the world of floriculture industry and the mechanism behind the onset of the very early events in the sequence still remains to be elucidated. Hence, for advancing the knowledge on the pertinent aspect of bud opening and senescence the literature has been cited under this review.     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals

In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.     

Expression of defender against apoptotic death (DAD-1) in Iris and Dianthus petals

The gene defender against apoptotic death ( DAD-1 ) prevents programmed cell death in animal cells. We investigated the expression pattern of DAD-1 in petals of iris ( Iris  ×  hollandica cv. Blue Magic) and carnation ( Dianthus caryophyllus cv. Etarro). DAD-1 expression in Iris petals was strongly reduced by the time of visible senescence, which occurs 4 days after flower opening. Microscopic analysis showed that most mesophyll cells had died prior to a clear decrease in DAD-1 expression and that epidermis cells started to die by that time. In carnation petals DAD-1 expression also decreased by the time of massive cell death. After ethylene treatment, DAD-1 expression in carnation again decreased concomitant with the advance in massive cell death. In conclusion, DAD-1 is not an early regulator of petal cell death. Its expression may be required for the programmed dismantling of cells, as it ceases only just prior to, or concomitant with, cell death.     

Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.     

Networking senescence-regulating pathways by using Arabidopsis enhancer trap lines

The last phase of leaf development, generally referred to as leaf senescence, is an integral part of plant development that involves massive programmed cell death. Due to a sharp decline of photosynthetic capacity in a leaf, senescence limits crop yield and forest plant biomass production. However, the biochemical components and regulatory mechanisms underlying leaf senescence are poorly characterized. Although several approaches such as differential cDNA screening, differential display, and cDNA subtraction have been employed to isolate senescence-associated genes (SAGs), only a limited number of SAGs have been identified, and information regarding the regulation of these genes is fragmentary. Here we report on the utilization of enhancer trap approach toward the identification and analysis of SAGs. We have developed a sensitive large-scale screening method and have screened 1,300 Arabidopsis enhancer trap lines and have identified 147 lines in which the reporter gene GUS (beta-glucuronidase) is expressed in senescing leaves but not in non-senescing ones. We have systematically analyzed the regulation of beta-glucuronidase expression in 125 lines (genetically, each contains single T-DNA insertion) by six senescence-promoting factors, namely abscisic acid, ethylene, jasmonic acid, brassinosteroid, darkness, and dehydration. This analysis not only reveals the complexity of the regulatory circuitry but also allows us to postulate the existence of a network of senescence-promoting pathways. We have also cloned three SAGs from randomly selected enhancer trap lines, demonstrating that reporter expression pattern reflects the expression pattern of the endogenous gene.