Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.     

Leaf senescence is accompanied by an early disruption of the microtubule network in Arabidopsis

The dynamic assembly and disassembly of microtubules (MTs) is essential for cell function. Although leaf senescence is a well-documented process, the role of the MT cytoskeleton during senescence in plants remains unknown. Here, we show that both natural leaf senescence and senescence of individually darkened Arabidopsis (Arabidopsis thaliana) leaves are accompanied by early degradation of the MT network in epidermis and mesophyll cells, whereas guard cells, which do not senesce, retain their MT network. Similarly, entirely darkened plants, which do not senesce, retain their MT network. While genes encoding the tubulin subunits and the bundling/stabilizing MT-associated proteins (MAPs) MAP65 and MAP70-1 were repressed in both natural senescence and dark-induced senescence, we found strong induction of the gene encoding the MT-destabilizing protein MAP18. However, induction of MAP18 gene expression was also observed in leaves from entirely darkened plants, showing that its expression is not sufficient to induce MT disassembly and is more likely to be part of a Ca(2+)-dependent signaling mechanism. Similarly, genes encoding the MT-severing protein katanin p60 and two of the four putative regulatory katanin p80s were repressed in the dark, but their expression did not correlate with degradation of the MT network during leaf senescence. Taken together, these results highlight the earliness of the degradation of the cortical MT array during leaf senescence and lead us to propose a model in which suppression of tubulin and MAP genes together with induction of MAP18 play key roles in MT disassembly during senescence.     

The impact of light intensity on shade-induced leaf senescence

Plants often have to cope with altered light conditions, which in leaves induce various physiological responses ranging from photosynthetic acclimation to leaf senescence. However, our knowledge of the regulatory pathways by which shade and darkness induce leaf senescence remains incomplete. To determine to what extent reduced light intensities regulate the induction of leaf senescence, we performed a functional comparison between Arabidopsis leaves subjected to a range of shading treatments. Individually covered leaves, which remained attached to the plant, were compared with respect to chlorophyll, protein, histology, expression of senescence-associated genes, capacity for photosynthesis and respiration, and light compensation point (LCP). Mild shading induced photosynthetic acclimation and resource partitioning, which, together with a decreased respiration, lowered the LCP. Leaf senescence was induced only under strong shade, coinciding with a negative carbon balance and independent of the red/far-red ratio. Interestingly, while senescence was significantly delayed at very low light compared with darkness, phytochrome A mutant plants showed enhanced chlorophyll degradation under all shading treatments except complete darkness. Taken together, our results suggest that the induction of leaf senescence during shading depends on the efficiency of carbon fixation, which in turn appears to be modulated via light receptors such as phytochrome A.     

The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature

Cold acclimation and over-wintering by herbaceous plants are energetically expensive and are dependent on functional plastid metabolism. To understand how the stroma and the lumen proteomes adapt to low temperatures, we have taken a proteomic approach (difference gel electrophoresis) to identify proteins that changed in abundance in Arabidopsis chloroplasts during cold shock (1 day), and short- (10 days) and long-term (40 days) acclimation to 5 degrees C. We show that cold shock (1 day) results in minimal change in the plastid proteomes, while short-term (10 days) acclimation results in major changes in the stromal but few changes in the lumen proteome. Long-term acclimation (40 days) results in modulation of the proteomes of both compartments, with new proteins appearing in the lumen and further modulations in protein abundance occurring in the stroma. We identify 43 differentially displayed proteins that participate in photosynthesis, other plastid metabolic functions, hormone biosynthesis and stress sensing and signal transduction. These findings not only provide new insights into the cold response and acclimation of Arabidopsis, but also demonstrate the importance of studying changes in protein abundance within the relevant cellular compartment.     

Presence of indole-3-acetic acid in chloroplasts ofNicotiana tabacum andPinus sylvestris

The compartmentation and metabolism of indole-3-acetic acid (IAA) was examined in protoplasts derived from needles ofPinus sylvestris L., leaves of normal plants ofNicotiana tabacum L., leaves ofN. tabacum plants carrying the T-DNA gene 1 (rG1 plants) and leaves ofN. tabacum plants carrying the T-DNA gene 2 (rG2 plants) by using a rapid cell-fractionation method. In all tissues, 30%–40% of the IAA pool was located in the chloroplast, while the remainder was found in the cytosol. Quantitative analysis of indole-3-ethanol (IEt) showed that in bothPinus andNicotiana the IEt pool was located exclusively in the cytosol. The only plant that contained endogenous indoleacetamide (IAAm) was therG1-mutant ofN. tabacum, expressing theAgrobacterium tumefaciens T-DNA gene 1. Cellular fractionation of protoplasts from this transgenic plant showed that the entire IAAm pool was located in the cytosol. Feeding experiments utilizing [5-³H]tryptophan, [5-³H]IEt, [1′-¹⁴C] and [2′-¹⁴C]IAA demonstrated that the biosynthesis and catabolism of IAA occurred in the cytosol in bothPinus and in the wild type and the different mutants ofNicotiana. Furthermore, the biosynthesis of IAAm in therG1 plants was also shown to be localized in the cytosol.     

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO₂ assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH₄ ⁺ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu.     

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

Malate dehydrogenase (MDH) catalyzes a reversible NAD⁺-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO₂ assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO₂ assimilation/intercellular CO₂ curves at low CO₂, and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.Plant tissues contain multiple isoforms of malate dehydrogenase (l-malate-NAD-oxidoreductase [MDH]; EC 1.1.1.37) that catalyze the interconversion of malate and oxaloacetate (OAA) coupled to reduction or oxidation of the NAD pool. These isoforms are encoded by separate genes in plants and have been shown to possess distinct kinetic properties as well as subcellular targeting and physiological functions (Gietl, 1992). While the MDH reaction is reversible, it strongly favors the reduction of OAA. The direction of the reaction in vivo depends on substrate/product ratios and the NAD redox state, and it can vary even in the same tissue due to prevailing physiological conditions. Isoforms operate in mitochondria, chloroplasts, peroxisomes, and the cytosol, but due to the ready transport and utilization of malate and OAA and the availability of NAD, this reaction can cooperate across compartments and is the basis for malate/OAA shuttling of reducing equivalents in many different metabolic schemes of plant cellular function (Krömer, 1995). It is clear, however, that the exchange through the membranes is strictly controlled, since large redox differences in NAD(H) pools exist between compartments (Igamberdiev and Gardeström, 2003).The mitochondrial MDH (mMDH) is thought to operate in at least three different pathways in plants. First, it is a classical tricarboxylic acid (TCA) cycle enzyme that oxidizes the malate product from the fumarase reaction to OAA for the citrate synthase-dependent condensation with acetyl-CoA to form citrate. Second, it is considered to operate in the reverse direction during the conversion of Gly to Ser by reducing OAA to malate and providing a supply of NAD⁺ for Gly decarboxylase (Journet et al., 1981). Third, in a more specialized pathway in C4 plants, it provides a supply of CO₂ for fixation in bundle sheath chloroplasts by reducing OAA (generated from Asp transported from mesophyll cells) into malate that is then decarboxylated by NAD-malic enzyme (NAD-ME) to CO₂ and pyruvate (Hatch and Osmond, 1976). Plant mitochondria can support TCA cycle activity with malate as the sole substrate due to MDH and NAD-ME, both ubiquitous in plants (Palmer, 1984). OAA is readily transported both into and out of isolated plant mitochondria (Douce and Bonner, 1972), in contrast to mammalian mitochondria, which are essentially impermeable to this organic acid.While these three mMDH schemes and metabolic schemes for other MDH isoforms are plausible, widely accepted, and consistent with a range of biochemical studies, the depletion, removal, and overexpression of specific MDH isoforms in plants have led to surprising insights into MDH roles in vivo. For example, the peroxisomal MDH (PMDH) was until recently generally considered to be involved in the synthesis of NADH for hydroxypyruvate reduction in the photorespiratory cycle and for the oxidation of NADH generated during β-oxidation of fatty acids, but its potential role in the oxidation of malate in the glyoxylate cycle was unclear. However, studies of the double knockout of PMDH in Arabidopsis (Arabidopsis thaliana) showed that while PMDH is essential for β-oxidation, its removal does not impair glyoxylate cycle activity (Pracharoenwattana et al., 2007) and has only a limited impact on hydroxypyruvate reduction (Cousins et al., 2008).Changes in mMDH have been reported both through the study of spontaneous mutants and the expression of antisense constructs. Spontaneous null mutants of mMDH1 in soybean (Glycine max) are linked to a yellow foliage phenotype and are associated with the removal of two of the three mMDH isoforms (Imsande et al., 2001). Expression of an antisense fragment of mMDH in tomato (Solanum lycopersicum), driven by the 35S promoter, lowered mMDH protein in mitochondria, decreased total cellular MDH by approximately 60%, but had a positive impact on photosynthetic activity, CO₂ assimilation rate, and total plant dry matter in long-day-grown plants (Nunes-Nesi et al., 2005). A range of carbohydrates also accumulated in the tomato antisense plants, as did redox-related compounds such as ascorbate. The increase in ascorbate content may be linked to the enhancement of photosynthesis, as ascorbate feeding to leaves can also increase photosynthetic performance (Nunes-Nesi et al., 2005). This link is not absolute, however, given that short-day-grown antisense tomato plants had stunted growth, which was potentially due to impaired photosynthesis, but still had elevated levels of ascorbate due to a higher ratio of reduction of the ascorbate pool compared with the wild type (Nunes-Nesi et al., 2008). Analysis of roots from these antisense tomato plants revealed a negative impact of mMDH loss, leading to a lower root dry weight and lower root respiratory rate (van der Merwe et al., 2009). This implies a distinct impact of mMDH loss on roots and shoots. Overexpression of cytosolic MDH led to a 4-fold elevation of root organic acids in alfalfa (Medicago sativa) plants and high rates of organic acid exudation that increased aluminum tolerance through metal chelation in the soil (Tesfaye et al., 2001). These studies imply that there is a complex form of functional redundancy between MDH isoforms in different compartments, allowing MDH in separate locations to maintain specific pathways via malate/OAA shuttling, or that a range of redox requirements that have been linked to MDH in accepted metabolic schemes are incorrect and other reactions couple NAD/NADH pool homeostasis. In addition, these studies clearly show that changes in the amount of MDH isoforms can alter metabolic flux into a range of organic acids and have far-reaching effects on plant growth and development.To better understand the importance of the mMDH and to determine if plants are viable without any mMDH isoforms due either to the role of NAD-ME and/or malate/OAA shuttling to other compartments, we have constructed and analyzed mMDH mutants in Arabidopsis. A major and a minor MDH isoform exist in Arabidopsis mitochondria, evidenced by differing levels of gene expression and differing protein abundance (Lee et al., 2008). We hypothesized that if mMDH works in concert with other MDH isoforms and is responsible for the reduction of OAA to malate for export from the mitochondrion, then if we remove mMDH, not only would the loss of extramitochondrial malate and the slowing of Gly decarboxylation limit photorespiratory carbon flux, but oxidation of NADH remaining in the mitochondrion could lead to elevated leaf respiration and alteration in plant growth. We found that not only did mutants have low photorespiratory flux, but they also increased respiration and had slow growth due to lowered net CO₂ assimilation. The previously established correlation between mMDH abundance, photosynthetic performance, and foliar ascorbate levels was also investigated. Elevated levels of the metabolite were found in Arabidopsis, consolidating the work done in tomato (Nunes-Nesi et al., 2005). Proteomic analyses, followed by immunodetection studies, unearthed altered abundance of the terminal enzyme of the ascorbate biosynthetic pathway, galactono-1,4-lactone dehydrogenase (GLDH), as a mechanistic element in the phenomenon linked directly to mitochondrial function.