Biosynthesis, targeting and processing of oleosin‐like proteins, which are major pollen coat components in Brassica napusMurphy, Denis J.; Ross, Joanne H. E.
doi: 10.1046/j.1365-313X.1998.00001.xpmid: N/A
The purpose of this study is to characterise the biosynthesis, targeting and processing of some of the major protein components of the pollen coat, or tryphine, of Brassica napus. The authors have N‐terminally sequenced 11 of the most abundant pollen coat polypeptides, and nine of these sequences correspond to proteolytically cleaved products of seven oleosin‐like genes, i.e. Oln B;1 to Oln B;6 and Oln B;11. The Oln B;11 gene product is co‐ or post‐translationally targeted in vitro to canine microsomal membranes. This implies that the oleosin‐like protein is targeted to the endoplasmic reticulum in tapetal cells in vivo. Affinity‐purified antibodies raised against a 20‐residue domain of Oln B;3 and B;4 gene products cross‐reacted with full‐length proteins of 45–48 kDa in early developing (< 2 mm to 5 mm) buds and anthers, but recognised truncated proteins of 32–38 kDa at later (4 mm to 7 mm) stages of development. The 45–48 kDa immunoreactive proteins were associated with a floating lipid body fraction obtained from a tapetal/locular fluid extract from maturing anthers and a major 48 kDa polypeptide from this fraction was confirmed by N‐terminal sequencing to be a full length product of the Oln B;3 gene. Quantitative immunocytochemical studies showed that the full length 45–48 kDa oleosin‐like proteins were specifically localised in the interior of tapetal cytoplasmic lipid bodies where they were associated with a regular hexagonal‐like fibrous reticulum. No significant labelling of elaioplasts was observed. The same antibodies specifically labelled 32–38 kDa oleosin‐like proteins on the extracellular pollen coat of maturing pollen grains. These results demonstrate for the first time that many of the major pollen coat proteins are derived from an endoproteolytic cleavage of precursor oleosin‐like proteins that originally accumulate within the large cytoplasmic lipid bodies of tapetal cells.
A pectate lyase from Zinnia elegans is auxin inducibleDomingo, Concha; Roberts, Keith; Stacey, Nicola J.; Connerton, Ian; Ruíz‐teran, Francisco; McCann, Maureen C.
doi: 10.1046/j.1365-313X.1998.00002.xpmid: 9680962
The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to reproducibly trans‐differentiate into tracheary elements (TE) after 96 h, while in the presence of auxin alone the cells simply elongate. In a search for genes involved in modifications to cell‐wall architecture before any overt signs of cell differentiation, a differential hybridization of a 72‐h cDNA library with probes from mRNA at time‐points of 24 h and 72 h was done revealing a number of transcripts up‐regulated between these times. One of these cDNAs shows homology to pectate lyase, a pectin‐degrading enzyme. The complete cDNA sequence (ZePel) corresponds to a translated protein of 44 kDa with an N‐terminal signal peptide of about 2 kDa, and one potential N‐glycosylation site. Northern analysis confirms that the strong expression of this gene during TE induction occurs at a very early stage of the process and is due solely to the presence of auxin in the induction medium. In situ hybridization studies in young Zinnia stems show that ZePel expression is associated with vascular bundles and shoot primordia. Recombinant protein made in Escherichia coli possesses calcium‐dependent pectate lyase activity. Pectate lyase activity is detected in elongating and differentiating in vitro cell populations. The role of this enzyme in remodelling the cell wall during cell elongation and differentiation is discussed.
Photosynthetic oxygen evolution within Sesbania rostrata stem nodulesJames, Euan K.; Minchin, Frank R. ; Oxborough, Kevin; Cookson, Alan; Baker, Neil R.; Witty, John F. ; Crawford, Robert M.M.; Sprent, Janet I.
doi: 10.1046/j.1365-313X.1998.00003.xpmid: N/A
The tropical wetland legume, Sesbania rostrata Brem. forms N2‐fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans. The N2‐fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non‐cyclic photosynthetic electron transport were concentrated within the mid‐ and inner‐cortex, close to the edge of the N2‐fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O2‐specific microelectrodes which showed that the concentration of O2 (pO2) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO2 of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow‐through acetylene reduction assay (no H2 evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO2 of 40%.
In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNADrager, Robert G.; Girard‐bascou, Jacqueline; Choquet, Yves; Kindle, Karen L.; Stern, David B.
doi: 10.1046/j.1365-313X.1998.00016.xpmid: 9680967
The acetate‐requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1–1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1–1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1–1 background is located in the 5′ untranslated region (UTR). However, when this 5′ UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1–1 background. Together, these results suggest that the 5′ end of the petD 5′ UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5′ UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5′→3′ exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5′ UTR protects the mRNA from 5′→3′ degradation in wild‐type cells.
A role for the vacuole in auxin‐mediated control of cytosolic pH by Vicia mesophyll and guard cellsFrohnmeyer, Hanns; Grabov, Alexander; Blatt, Michael R.
doi: 10.1046/j.1365-313X.1998.00013.xpmid: N/A
A role for cytosolic pH (pHi) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pHi are a common consequence of hormonal stimuli in plant cells and contribute to hormonally evoked ion channel control, the origins of these changes remain unknown. To examine a possible role for the tonoplast and vacuolar compartment in these events, pHi was measured in the presence of auxins and during cytosolic H+ loading with weak acid in vacuolate and evacuolate protoplasts, both from mesophyll and guard cells of Vicia faba L. Evacuolate protoplasts were obtained following ultracentrifugation on Percoll gradients, and pHi of single protoplasts was recorded in both vacuolate and evacuolate preparations using fluorescence ratio microphotometry and the pH‐sensitive dye BCECF. External pH measurements indicated a roughly twofold increase in the rate of net H+ secretion in evacuolate compared with vacuolate protoplasts, and showed that evacuolate protoplasts retained the characteristic stimulation of H+ secretion in the presence of auxin. BCECF fluorescence recording gave resting pHi values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pHi were evoked in vacuolate protoplasts by 10 μM concentrations of the auxins 1‐naphthalene acetic acid and 3‐indoyl‐acetic acid, and not by the inactive (anti‐auxin) analogue 2‐naphthalene‐acetic acid. However, auxin treatments failed to evoke a change in pHi in all but one of 12 experiments with evacuolate protoplasts. Evacuolation also appeared to reduce the transient, dynamic H+ buffering capacity of the protoplasts in the face of acid pHi loads imposed by adding Na+‐butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short‐term pHi homeostasis and generation of hormonally evoked H+ signalling in plant cells; they also conform with the view that the decrease in pHiper se is not a primary determinant in the stimulation of H+ secretion by auxin.