Susheng Gan

Inhibition of leaf senescence by autoregulated production of cytokinin

Controlling expression of IPT, a gene encoding isopentenyl transferase (the enzyme that catalyzes the rate-limiting step in cytokinin biosynthesis), with a senescence-specific promoter results in the suppression of leaf senescence. Transgenic tobacco plants expressing this chimeric gene do not exhibit the developmental abnormalities usually associated with IPT expression because the system is autoregulatory. Because sufficient cytokinin is produced to retard senescence, the activity of the senescence-specific promoter is attenuated. Senescence-retarded leaves exhibit a prolonged, photosynthetically active life-span. This result demonstrates that endogenously produced cytokinin can regulate senescence and provides a system to specifically manipulate the senescence program.

Making Sense of Senescence (Molecular Genetic Regulation and Manipulation of Leaf Senescence)

Leaf senescence is the final stage of leaf development. In forests of deciduous trees, the autumn colors that develop during leaf senescence are of great aesthetic value. This process is also of great practical value because during leaf senescence, nutrients are recycled to other parts of the plant. For example, nitrogen from leaves of deciduous trees is used for the synthesis of storage proteins in stems that will support growth during the following spring (Clausen and Apel, 1991). However, in an agricultural setting, leaf senescence may limit yield in certain crops. Senescence also contributes to the postharvest loss of vegetable crops. Therefore, studying leaf senescence will not only contribute to our knowledge about this fundamental developmental process, but may also lead to ways of manipulating senescence for agricultural applications. There have been many physiological, biochemical, and molecular studies of leaf senescence. These studies show that during senescence leaf cells undergo highly coordinated changes in cell structure, metabolism, and gene expression. The earliest and most significant change in cell structure is the breakdown of the chloroplast, the organelle that contains up to 70% of the leaf protein. Metabolically, carbon assimilation (photosynthesis) is replaced by catabolism of chlorophyll and macromolecules such as proteins, membrane lipids, and RNA so that some of the released nutrients can be recycled. At the molecular level, these changes are accompanied by, or perhaps driven by, changes in gene expression. In this Update, we summarize physiological and biochemical studies that have contributed to the present understanding of leaf senescence, then we discuss current molecular investigations into the regulatory mechanism(s) underlying leaf senescence, and, finally, we review some molecular approaches toward the manipulation of leaf senescence.

A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment

The expression of several Arabidopsis thaliana senescence-associated genes (SAGs) in attached and/or detached leaves was compared in response to age, dehydration, darkness, abscisic acid, cytokinin, and ethylene treatments. Most of the SAGs responded to most of the treatments in a similar fashion. Detachment in darkness and ethylene were the strongest inducers of both SAGs and visible yellowing. Detachment in light was also a strong inducer of SAGs, but not of visible yellowing. The other treatments varied more in their effects on individual SAGs. Responses were examined in both older and younger leaves, and generally were much stronger in the older ones. Individual SAGs differed from the norms in different ways, however, suggesting that their gene products play a role in overlapping but not identical circumstances. Some SAGs responded quickly to treatments, which may indicate a direct response. Others responded more slowly, which may indicate an indirect response via treatment-induced senescence. Four new SAGs were isolated as part of this work, one of which shows strong similarity to late embryogenesis-abundant (Lea) genes.

Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence

In this work, the role of jasmonic acid (JA) in leaf senescence is examined. Exogenous application of JA caused premature senescence in attached and detached leaves in wild-type Arabidopsis but failed to induce precocious senescence of JA-insensitive mutantcoi1 plants, suggesting that the JA-signaling pathway is required for JA to promote leaf senescence. JA levels in senescing leaves are 4-fold higher than in non-senescing ones. Concurrent with the increase in JA level in senescing leaves, genes encoding the enzymes that catalyze most of the reactions of the JA biosynthetic pathway are differentially activated during leaf senescence in Arabidopsis, except for allene oxide synthase, which is constitutively and highly expressed throughout leaf development. Arabidopsis lipoxygenase 1 (cytoplasmic) expression is greatly increased but lipoxygenase 2 (plastidial) expression is sharply reduced during leaf senescence. Similarly,AOC1 (allene oxide cyclase 1),AOC2, and AOC3 are all up-regulated, whereas AOC4 is down-regulated with the progression of leaf senescence. The transcript levels of 12-oxo-PDA reductase 1 and 12-oxo-PDA reductase 3 also increase in senescing leaves, as does PED1 (encoding a 3-keto-acyl-thiolase for β-oxidation). This represents the first report, to our knowledge, of an increase in JA levels and expression of oxylipin genes during leaf senescence, and indicates that JA may play a role in the senescence program.

Markers for hypersensitive response and senescence show distinct patterns of expression.

Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.

A Gene Encoding an Acyl Hydrolase Is Involved in Leaf Senescence in Arabidopsis

SAG101, a leaf senescence-associated gene, was cloned from an Arabidopsis leaf senescence enhancer trap line and functionally characterized. Reporter gene and RNA gel blot analyses revealed that SAG101 was not expressed until the onset of senescence in leaves. A recombinant SAG101 fusion protein overexpressed in Escherichia coli displayed acyl hydrolase activity. Antisense RNA interference in transgenic plants delayed the onset of leaf senescence for ∼4 days. Chemically induced overexpression of SAG101 caused precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants. These data suggest that SAG101 plays a significant role in leaf senescence.

Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance

Trichome glands on the surface of many higher plants produce and secrete exudates affecting insects, microbes, and herbivores. Metabolic engineering of gland exudation has potential for improving pest/disease resistance, and for facilitating molecular farming. We identified a cytochrome P450 hydroxylase gene specific to the trichome gland and used both antisense and sense co-suppression strategies to investigate its function. P450-suppressed transgenic tobacco plants showed a ≥41% decrease in the predominant exudate component, cembratriene-diol (CBT-diol), and a ≥19-fold increase in its precursor, cembratriene-ol (CBT-ol). Thus, the level of CBT-ol was raised from 0.2 to ≥4.3% of leaf dry weight. Exudate from antisense-expressing plants had higher aphidicidal activity, and transgenic plants with exudate containing high concentrations of CBT-ol showed greatly diminished aphid colonization responses. Our results demonstrate the feasibility of significantly modifying the natural-product chemical composition and aphid-interactive properties of gland exudates using metabolic engineering. The results also have implications for molecular farming.

Bidirectionalization of polar promoters in plants

A typical eukaryotic promoter consists of a minimal promoter and other upstream cis elements. The minimal promoter is essentially a TATA box region where RNA polymerase II, TATA-binding protein (TBP), and TBP-associated factors (TAFs) bind to initiate transcription, but minimal promoters alone have no transcriptional activity. The cis elements, to which tissue-specific or development-specific transcription factors bind, individually or in combination, determine the spatio-temporal expression pattern of a promoter at the transcriptional level. The arrangement of upstream cis elements followed by a minimal promoter sets the polarity of the promoter. Promoters in plants that have been cloned and widely used for both basic research and biotechnological application are generally unidirectional, directing only one gene that has been fused at its 3′ end (downstream). It is often necessary to introduce multiple genes into plants for metabolic engineering and trait stacking. It is also desirable to minimize or avoid repeated use of a single promoter that may cause transcriptional gene silencing. Here we describe a strategy to make polar promoters bidirectional so that one promoter can direct the expression of two genes, one on each end of the promoter.

Identical promoter elements are involved in regulation of the OPR1 gene by senescence and jasmonic acid in Arabidopsis

Like other developmental processes, the terminal phase of leaf development, generally referred to as leaf senescence, regulates a subset of genes whose transcript abundances are increased during senescence. Jasmonic acid (JA), a plant growth regulator, also regulates the expression of subsets of genes in many aspects of plant growth and development, including leaf senescence. However, the underlying molecular mechanisms by which senescence and JA modulate gene expression are poorly understood. During an effort to isolate senescence-associated genes, we identified an Arabidopsis enhancer trap line in which the reporter gene GUS is up-regulated by both senescence and JA. The T-DNA tagged gene was subsequently cloned using thermal asymmetric interlaced PCR (TAIL-PCR). This gene encodes a 12-oxo-phytodienoic acid-10,11-reductase (OPR1). Consistent with the GUS expression data, RNA gel blot analysis showed that OPR1 was indeed up-regulated by both senescence and JA. Promoter deletion analysis and linker-scanning mutagenesis assays were employed to unveil the molecular bases of OPR1 regulation by senescence and JA. Two regulatory cis elements, namely JASE1 (5′-CGTCAATGAA-3′) and JASE2 (5′-CATACGTCGTCAA-3′), in the promoter region of the gene, were identified. While JASE2 contains a mixed A/C box-like motif, JASE1 represents a new motif without any signature sequence so far reported. Both elements were required for the up-regulation of OPR1 by leaf senescence and JA, suggesting that leaf senescence and JA may share a common molecular mechanism for modulating OPR1.

A novel zinc-finger protein with a proline-rich domain mediates ABA-regulated seed dormancy in Arabidopsis.

Seed dormancy is an important developmental process that prevents pre-harvest sprouting in many grains and other seeds. Abscisic acid (ABA), a plant hormone, plays a crucial role in regulating dormancy but the underlying molecular regulatory mechanisms are not fully understood. An Arabidopsis zinc-finger gene, MEDIATOR OF ABA-REGULATED DORMANCY 1(MARD1) was identified and functionally analyzed. MARD1expression is up-regulated by ABA. A T-DNA insertion in the promoter region downstream of two ABA-responsive elements (ABREs) renders MARD1unable to respond to ABA. The mard1 seeds are less dormant and germinate in total darkness; their germination is resistant to external ABA at the stage of radicle protrusion. These results suggest that this novel zinc-finger protein with a proline-rich N-terminus is an important downstream component of the ABA signaling pathway that mediates ABA-regulated seed dormancy in Arabidopsis.