Functions of the water soluble chlorophyll-binding protein in plants

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700-900 μmol photons m⁻² s⁻¹. These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.     

Chlorophyllase versus pheophytinase as candidates for chlorophyll dephytilation during senescence of broccoli

Degradation of chlorophylls during senescence is a highly regulated process which requires the concerted action of several enzymes. Traditionally, it has been stated that the dismantling process of the chlorophyll molecule begins with a dephytilation step, followed by Mg²⁺ removal and other breakdown reactions. Recently, new evidence suggests the possibility of a rearrangement in the first two steps of this process, occurring Mg²⁺ removal prior to the loss of the phytol side chain. With the purpose of approximating to the real sequential order of these reactions and to assess if dephytilation occurs on intact (catalyzed by chlorophyllase) or Mg-free (catalyzed by pheophytinase) chlorophyll, expression of both genes was analyzed in broccoli tissue during senescence. Samples of broccoli florets treated with plant hormones, such as cytokinin and ethylene were utilized, as to assess the effect of such compounds on the expression of these genes. Results showed that chlorophyllase expression did not correlate to typical expression patterns for genes related to senescence, since a decrease in expression during senescence was found for one of the two chlorophyllase genes analyzed, and the hormonal-treatment effects on gene expression did not match those observed on chlorophyll content for both chlorophyllase genes. Pheophytinase expression patterns, on the other hand, displayed an increase in the first 3 days of induced senescence, followed by lower expression values towards the end of the experiment. Samples subjected to postharvest treatments mostly showed an inhibition of pheophytinase expression, especially in samples in which degradation of chlorophylls had been delayed. These results suggest that pheophytinase expression correlates to the visual manifestation of postharvest treatments, supporting the possibility that this enzyme is responsible for the dephytilation step in chlorophyll breakdown.     

Characterization of sol-gel entrapped chlorophyllase

Immobilization of membrane proteins remains a challenge compared to soluble proteins. The membrane protein-chlorophyllase was successful entrapped in tetramethoxysilane (TMOS)-based sol-gel in the presence of lipid. Activity was examined against mixing rate, incubation temperature, time, substrate, acetone, and canola oil concentration. The external mass transfer of chlorophyll is not the rate-limiting step at higher mixing rates. Stability against temperature and acetone as denaturant was enhanced. In spite of the fact that an initial reaction lag phase was observed, 20% more chlorophyll was hydrolyzed, compared to reaction with free enzyme by the end of a 12 h assay. The initial lower activity demonstrated by entrapped chlorophyllase is likely due to the diffusion resistance of chlorophyll into and within the entrapment matrix. This hypothesis was substantiated by a low diffusion coefficient on the order of 10(-14) m(2)/s obtained for chlorophyll in nanoporous sol-gel particles. Pore size distribution of nanoporous wet TMOS-based sol-gel with or without protein was determined by thermoporometry. The change in pore morphology upon doping with chlorophyllase suggests that protein acts as a template during the sol-gel process.     

The role of chlorophyllase in degreening canola (Brassica napus) seeds and its activation by sublethal freezing

Chlorophyllase mediates dephytylation of chlorophylls and pheophytins during seed degreening in canola ( Brassica napus cv. Westar). Degreening can be correlated with chlorophyllase activity in vitro, but it is difficult to demonstrate in vivo activity because of low levels of the dephytylated breakdown products during rapid degreening. If, however, degreening is inhibited by sublethal freezing, chlorophyllide and pheophorbide accumulation can be related to the action of chlorophyllase. Changes in the rate of in vitro dephytylation during degreening and the dramatic increase following freezing may indicate enzyme activation and de novo enzyme synthesis. Evidence from Western blots is presented in support of de novo synthesis. It is concluded that failure of the seed to degreen following sublethal freezing does not result from a reduction in chlorophyllase activity.     

Dynamic change in photosynthetic pigments and chlorophyll degradation elicited by cereal aphid feeding

The concentration of photosynthetic pigments (i.e., chlorophylls a and b, and carotenoids) and chlorophyll degradation enzyme (i.e., chlorophyllase, oxidative bleaching, and Mg-dechelatase) activities on aphid-damaged and non-damaged regions of the infested leaves were determined with two infestation periods (6 and 12 days). Russian wheat aphid [Diuraphis noxia (Mordvilko) (Hemiptera: Aphididae)] feeding caused significant losses of chlorophylls a and b and carotenoids in the damaged regions. However, bird cherry-oat aphid [Rhopalosiphum padi (L.) (Hemiptera: Aphididae)] feeding did not, except a significantly lower level of carotenoids was observed in the damaged regions from the short-infestation (6-day) samples. Interestingly, the non-damaged regions of D. noxia-infested leaves on both sampling dates had a significant increase of chlorophylls a and b and carotenoid concentrations when compared with the uninfested leaves. Although D. noxia feeding did not cause any changes in either chlorophyll a/b or chlorophyll (a+b)/carotenoid ratio between the damaged and non-damaged leaf regions on short-infestation (6-day) samples, a significantly lower chlorophyll a/b ratio was detected in long-infestation (12-day) samples. The assays of chlorophyllase and oxidative bleaching activities showed no significant differences between the damaged and non-damaged regions of the infested leaves on either sampling date. Mg-dechelatase activity, however, was significantly higher in D. noxia-damaged than non-damaged leaf regions from the short-infestation samples, while no differences were detected from the long-infestation samples. Furthermore, the long-infestation samples showed that Mg-dechelatase activity from both D. noxia-damaged and non-damaged regions increased significantly in comparison with the respective regions of either uninfested or R. padi-infested leaves. We infer that non-damaged regions of D. noxia-infested leaves compensate for the pigment losses in the damaged regions, and that Mg-dechelatase activity changed dynamically from a localized response to a systemic response as infestation duration extends. The findings from this study on cereal aphid-elicited chlorosis (or desistance) would help us to elucidate plant resistance mechanisms, in particular plant tolerance to non-defoliating herbivory.     

Effects of aluminium on photosynthetic performance in Al-sensitive and Al-tolerant maize inbred lines

Maize plant inbred lines, one Al-sensitive (B-73) and two Al-tolerant (F-2 and L-2039), were grown hydroponically in the presence of 200 μM Al. After 13 d of growth, root and shoot lengths, photosystem 2 (PS2) activity, chlorophyll (Chl) content, 5-aminolevulinic acid (5-ALA) synthesis rate, chlorophyllase (Chlase) activity, and N, Mg, Fe, and Mn contents in leaves were determined. PS2 activity and Chl content were most severely affected by Al in B-73, but F-2 was almost unaffected. This was in accordance with Al-accumulation in the plants. The observed changes in B-73 coincided with 5-ALA synthesis inhibition, Chlase activation, and leaf deprivation of Fe and Mg. In Al-treated L-2039 plants, the leaf Mg and Mn contents were decreased. Also, an excessive Chlase activation was found in Al-treated L-2039, without a substantial Chl loss. This may indicate the activation of different enzyme pools in tolerant and sensitive genotypes under low-stress conditions.     

In vitro enzymatic chlorophyll catabolism in wheat elicited by cereal aphid feeding

Chlorophyll degradation is a complex phenomenon that often accompanies insect feeding damage to plants. Loss of chlorophyll can be initiated by several reactions, including oxidative bleaching, chlorophyllase activity, and Mg-dechelatase activity. Extracts from the Russian wheat aphid [Diuraphis noxia (Mordvilko)], the bird cherry-oat aphid [Rhopalosiphum padi (L.)], and aphid-infested and uninfested wheat plants were assayed in vitro for activities involved in chlorophyll degradation. Although the initial infestation was the same (10 apterous adults) for both aphid species, D. noxia weight was significantly higher than R. padi after feeding for 12 days. Consequently, D. noxia feeding caused greater fresh leaf weight reduction than R. padi feeding. Chlorophyll degradation assays showed no activity from either D. noxia or R. padi extracts. Plant extract assays showed a significant difference in Mg-dechelatase activity, while no difference was detected in either the chlorophyllase or oxidative bleaching pathways among the aphid-infested or uninfested plant extracts. Diuraphis noxia-infested leaf extracts showed a greater increase of Mg-dechelatase activity than either R. padi-infested or the uninfested plants. The findings suggest that leaf chlorosis elicited by D. noxia feeding is different from the chlorophyll degradation that occurs in natural plant senescence. Aphid-elicited chlorosis might be the result of a Mg-dechelatase-driven catabolism of chlorophyll in challenged wheat seedlings, however, the factor(s) from D. noxia that elicited the increase of Mg-dechelatase activity still remain to be determined.     

AtCLH2, a Typical but Possibly Distinctive Chiorophyllase Gene in Arabidopsis

Chlorophyllase (EC 3.1.1.14) is involved in the first step of chlorophyll degradation. Isolation of chlorophyllase genes greatly facilitates characterization of chlorophyllase properties and elucidation of molecular regulation of their in vivo activities. There are two chlorophyllase genes, AtCLH1 and AtCLH2, in Arabidopsis thallana. The in vivo roles of AtCLH1 have been reported previously. However, few studies have been carried out on AtCLH2. Here,we show that purified recombinant Chlase2, encoded by AtCLH2, exhibits in vitro chlorophyllase activity. Interestingly,"activation" of in vitro activity of the recombinant Chlase2 required higher concentrations of a detergent or a polar solvent. To determine its activity in vivo, the expression of AtCLH2 was inhibited by RNA interference. RNAi plants showed decreased contents of chlorophyllide without a substantial change in the total amount of the extractable chlorophyll and consequently presented lower chlorophyllide to chlorophyll ratios in their leaves. In addition, the two AtCLHs exhibited differential expression patterns. Our results suggest that AtCLH2 might play a distinctive role in chlorophyll catabolism in vivo.