Marcelo Ehlers Loureiro

Enhanced Photosynthetic Performance and Growth as a Consequence of Decreasing Mitochondrial Malate Dehydrogenase Activity in Transgenic Tomato Plants

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the mitochondrial malate dehydrogenase gene in the antisense orientation and exhibiting reduced activity of this isoform of malate dehydrogenase show enhanced photosynthetic activity and aerial growth under atmospheric conditions (360 ppm CO2). In comparison to wild-type plants, carbon dioxide assimilation rates and total plant dry matter were up to 11% and 19% enhanced in the transgenics, when assessed on a whole-plant basis. Accumulation of carbohydrates and redox-related compounds such as ascorbate was also markedly elevated in the transgenics. Also increased in the transgenic plants was the capacity to use l-galactono-lactone, the terminal precursor of ascorbate biosynthesis, as a respiratory substrate. Experiments in which ascorbate was fed to isolated leaf discs also resulted in increased rates of photosynthesis providing strong indication for an ascorbate-mediated link between the energy-generating processes of respiration and photosynthesis. This report thus shows that the repression of this mitochondrially localized enzyme improves both carbon assimilation and aerial growth in a crop species.

Photochemical responses and oxidative stress in two clones of Coffea canephora under water deficit conditions

Abstract The effects of water deficit on photochemical parameters and activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as, cellular damages were investigated in two clones of Coffea canephora differing in drought tolerance. After 6 days without irrigation, predawn leaf water potential fell to −3.0 MPa that was accompanied by the suppression of net photosynthesis in both clones. The variable to maximum chlorophyll fluorescence ratio remained unchanged regardless of the imposed treatments. Both clones showed a similar decline (about 25%) in the photochemical quenching coefficient, but only the drought-sensitive clone exhibited an enhancement (31%) of thermal deactivation under water deficit conditions. The quantum yield of electron transport decreased similarly in both genotypes. Under drought conditions, activities of SOD, CAT and APX increased to a greater extent in the drought-tolerant clone than in the drought-sensitive one. This seemed to be matched with higher protection against oxidative stress, as judged from the lower levels of lipid peroxidation and electrolyte leakage in the drought-tolerant clone. Thus, the ability to increase the antioxidant system activity in order to limit cellular damages might be an important attribute linked to the drought tolerance in C. canephora.

Reduced Expression of Aconitase Results in an Enhanced Rate of Photosynthesis and Marked Shifts in Carbon Partitioning in Illuminated Leaves of Wild Species Tomato

Wild species tomato (Lycopersicon pennellii) plants bearing a genetic lesion in the gene encoding aconitase (Aco-1; aconitate hydratase EC 4.2.1.3) were characterized at molecular and biochemical levels. The genetic basis of this lesion was revealed by cloning the wild-type and mutant alleles. The mutation resulted in lowered expression of the Aco-1 transcript and lowered levels of both cytosolic and mitochondrial aconitase protein and activity. After in silico analysis, we concluded that in the absence of a recognizable target sequence, the best explanation for the dual location of this protein is inefficient targeting. Biochemical analysis of leaves of the Aco-1 accession suggested that they exhibited a restricted flux through the Krebs cycle and reduced levels of Krebs cycle intermediates but were characterized by elevated adenylate levels and an enhanced rate of CO2 assimilation. Furthermore, the analysis of both steady-state metabolite levels and metabolic fluxes revealed that this accession also exhibited elevated rates of photosynthetic Suc synthesis and a corresponding increase in fruit yield. Therefore, we conclude that the Krebs cycle normally competes with the Suc synthetic pathway for carbon but is not essential for the supply of energy to fuel the operation of this pathway.

Differential responses of superoxide dismutase in freezing resistant Solanum curtilobum and freezing sensitive Solanum tuberosum subjected to oxidative and water stress

In photosynthetic tissues superoxide dismutase (SOD) plays an important role by scavenging the superoxide radical whose production is an usual reaction in chloroplast thylakoids. To test the differential response of SOD, two Andean potato species differing in frost resistance, Solanum curtilobum (frost resistant) and Solanum tuberosum (frost sensitive), were subjected to methyl viologen-mediated oxidative stress and polyethylene glycol (PEG)-induced water stress. A significant increment (approximately two-fold) in total SOD and FeSOD activity, which occupied about 50% of the total activity, was found when leaves of S. curtilobum were exposed to water stress. In contrast, the SOD activity in leaves of S. tuberosum remained unchanged. The exposure of leaves of S. curtilobum to oxidative stress increased total SOD and FeSOD activity by 350%. High correlation between SOD activity and the F(v)/F(m )ratio under both PEG induced water stress and MV-mediated oxidative stress was observed. This suggests that SOD can protect PSII from superoxide generated by oxidative and water stress. The higher SOD activity could be an important mechanism to explain why some natives Andean potato like S. curtilobum are more resistant to abiotic stresses than S. tuberosum.

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco.

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

Drought tolerance of two field-grown clones of Coffea canephora

We compared tolerance to soil drought of two field-grown clones of Coffea canephora (clone 46, drought-sensitive; and clone 120, drought-tolerant). Under irrigation, there were no marked differences between the clones in water relation parameters, gas exchange and total leaf area. Under rainfed conditions, clone 46 showed osmotic adjustment and increased tissue rigidity. These adjustments, however, were incapable of preventing substantial decreases in xylem pressure potential. By contrast, clone 120 did not exhibit osmotic adjustment, but was able to increase tissue elasticity and to maintain xylem pressure potentials to a greater extent than clone 46 (despite having twice the total leaf area of this clone). Stomatal conductance was lowered by drought in clone 120 but not in clone 46. Carbon assimilation per unit leaf area in both clones remained unaffected under stress. Long-term water use efficiency (WUE), as estimated through carbon isotope discrimination, was consistently greater in clone 120 than in clone 46. Because of these traits, clone 120 was better able to postpone dehydration and to maintain whole-tree photosynthesis. It is proposed that these features should decisively contribute to buffer its productivity in drought-prone areas.

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

Limitations to photosynthesis inCoffea canephoraas a result of nitrogen and water availability

Summary Plants of C. canephora grown in pots under low nitrogen (LN) or high nitrogen (HN) applications were submitted to either cyclic water stress or daily irrigation. Water deficit led to marked decreases in net carbon assimilation rate (A) and, to a lesser extent, in stomatal conductance (gs), regardless of the N treatments. In well-watered plants, A appreciably increased in HN plants relative to LN plants without significant changes in gs. As a whole, changes in internal CO2 concentration predominantly reflected changes in A rather than in gs. Under irrigated conditions, A, but not gs, correlated with leaf N concentration in a curvilinear way. Photosynthetic nitrogen-use efficiency was considerably low, and decreased with increasing leaf N concentration. Limited N, but not water, slightly decreased the maximum photochemical efficiency of photosystem II (PSII). Under continuous irrigation, LN plants had a smaller quantum yield of electron transport (ϕPSII) through slight decreases of photochemical quenching (qp) and capture efficiency of excitation energy by open PSII reaction centres, and increases in Stern-Volmer non-photochemical quenching. Under water-stressed conditions, changes in PSII photochemistry were apparent only in HN plants, with a 25 % decrease in ϕPSII, due mainly to variations in qp. Biochemical constraints, rather than stomatal or photochemical limitations, provoked the decreases in A under limited supply of either N or water.

Growth and photosynthetic down-regulation in Coffea arabica in response to restricted root volume

Coffee (Coffea arabica L.) plants were grown in small (3-L), medium (10-L) and large (24-L) pots for 115 or 165 d after transplanting (DAT), which allowed different degrees of root restriction. Effects of altered source : sink ratio were evaluated in order to explore possible stomatal and non-stomatal mechanisms of photosynthetic down-regulation. Increasing root restriction brought about large and general reductions in plant growth associated with a rising root : shoot ratio. Treatments did not affect leaf water potential or leaf nutrient status, with the exception of N content, which dropped significantly with increasing root restriction even though an adequate N supply was available. Photosynthesis was severely reduced when plants were grown in small pots; this was largely associated with non-stomatal factors, such as decreased Rubisco activity. At 165 DAT contents of hexose, sucrose, and amino acids decreased in plants grown in smaller pots, while those of starch and hexose-P increased in plants grown in smaller pots. Photosynthetic rates were negatively correlated with the ratio of hexose to free amino acids, but not with hexose content. Activities of acid invertase, sucrose synthase, sucrose-P synthase, fructose-1,6-bisphosphatase, ADP-glucose pyrophosphorylase, starch phosphorylase, glyceraldehyde-3-P dehydrogenase, PPi : fructose-6-P 1-phosphotransferase and NADP : glyceraldehyde-3-P dehydrogenase all decreased with severe root restriction. Glycerate-3-P : Pi and glucose-6-P : fructose-6-P ratios decreased accordingly. Photosynthetic down-regulation was unlikely to have been associated directly with an end-product limitation, but rather with decreases in Rubisco. Such a down-regulation was largely a result of N deficiency caused by growing coffee plants in small pots.

Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems.

The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5′ flanking sequences were fused to β-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (−358 to −211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (−211 to −80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter.