ABSTRACTThe effect of K+ deficiency on the plasma membrane (PM) H+‐ATPase was studied in young stems of poplar plants (Populus tremula × tremuloides) grown with low or full‐strength K+ supply. Immunological assays using different antibodies were applied to test if K+ deficiency affects the amount of immunodetectable PM H+‐ATPases in the stem tissue. The monoclonal antibody clone 46 E5 B11 revealed an increased abundance of PM H+‐ATPases under conditions of low K+ supply, and immunolabelling experiments showed that this increase was restricted to vessel‐associated cells (VACs) of the wood ray parenchyma. Replacement of the monoclonal antibody by a polyclonal antibody against PM H+‐ATPase gave a specific immunoreactivity on blots as well as tissue sections too, but the labelling intensity showed no difference between plants with low or full‐strength K+ supply. Measurements of extracellular H+ concentrations using non‐invasive, H+‐selective microelectrodes revealed a lowering of the pH at the surface of VACs and an enhancement of net efflux of H+ in plants grown with low K+ supply. The present results indicate an up‐regulation of specific isoforms of the PM H+‐ATPase in VACs under K+‐deficient conditions and suggest a key role for these PM H+‐ATPases in unloading K+ from the xylem stream.

To study the involvement of potassium in wood formation, poplar plants ( Populus tremula L. x Populus tremuloides Michx.) were grown over a period of one growing season, under different potassium regimes. Seasonal changes in cambial potassium content, osmotic potential, and cambial activity correlated strongly throughout the season, increasing from spring to summer and decreasing from summer to autumn. Moreover, changing the potassium supply during the growing season affected the seasonal changes of these parameters in a similar way. Low potassium supply markedly reduced cambial activity, the number of expanding cambial cell derivatives, the seasonal rate of radial wood increment, and the vessel frequency. The possible effect of hormones on potassium-dependent cambial growth was investigated and revealed that abscisic acid (ABA) strongly decreased the potassium content within the cambial zone and reduced cambial activity, as well as the number of expanding cambial cell derivatives. In summary, our results indicate a key role for potassium in the regulation of cambial growth and wood formation due to its strong impact on osmoregulation in expanding cambial cells. They also demonstrate involvement of ABA in regulation of potassium-dependent cambial growth.

The cambial K+ content of poplar increases during the growth period in a K+ supply dependent manner. Upon K+ starvation or application of tetraethylammoniumchloride (TEA+), a K+ channel blocker, the average vessel lumen and expansion zone area were significantly reduced. In search for the molecular basis of potassium-dependent xylogenesis in poplar, K+ transporters homologous to those of known function in Arabidopis phloem- and xylem-physiology were isolated from a poplar wood EST library. The expression profile of three distinct K+ channel types and one K+ transporter, Populus tremula K+ uptake transporter 1 (PtKUP1), was analysed by quantitative RT-PCR. Thereby, we found P. tremula outward rectifying K+ channel (PTORK) and P. tremula K+ channel 2 (PTK2) correlated with the seasonal wood production. K+ transporter P. tremula 1 (KPT1) was predominantly found in guard cells. Following the heterologous expression in Xenopus oocytes the biophysical properties of the different channels were determined. PTORK, upon membrane de-polarization mediates potassium release. PTK2 is almost voltage independent, carrying inward K+ flux at hyperpolarized potential and K+ release upon de-polarization. PtKUP1 was expressed in a K+ uptake-deficient Escherichia coli strain, where this K+ transporter rescued K+-dependent growth. In order to link the different K+ transporters to the cambial activity and wood production, we compared the expression profiles to seasonal changes in the K+ content of the bark as well as xylem vessel diameter. Thereby, we found PTORK and PTK2 transcripts to follow the annual K+ variations in poplar branches. PtKUP1 was expressed at a low level throughout the year, suggesting a housekeeping function. From these data, we conclude that K+ channels are involved in the regulation of K+-dependent wood production.

Subunit monomers and oligomers of crystalloid-type legumins are major components of SDS-soluble fractions from Metasequoia glyptostroboides (Dawn redwood, Taxodiaceae) seed proteins. The subunits are made up of disulfide linked alpha-polypeptides and beta-polypeptides with molecular masses of 33 kDa and 23-25 kDa, respectively. Unusually for legumins, those from Metasequoia are glycosylated and the carbohydrate moieties are residing in the C-terminal region of the respective beta-polypeptides. A Metasequoia endosperm cDNA library has been constructed and legumin-encoding transcripts representing two divergent gene subfamilies have been characterized. Intersubfamily comparisons reveal 75% identity at the amino acid level and the values range from 53-35% when the legumin precursors deduced were compared with those from angiosperms. The predicted sequences together with data from amino acid sequencing prove that post-translational processing of Metasequoia prolegumins is directed to two different processing sites, each of them specific for one of the legumin subfamilies. The sites involved differ in their relative position and in the junction to be cleaved: Metasequoia legumin precursors MgLeg18 and MgLeg26 contain the conventional post-translational Asn-Gly processing site, which is generally regarded as highly conserved. In contrast, the MgLeg4 precursor is lacking this site and post-translational cleavage is directed to an unusual Asn-Thr processing site located in its hypervariable region, causing N-terminal extension of the beta-polypeptide relative to those hitherto known. Evidence is given that the unusual variant of processing also occurs in other conifers. Phylogenetic analysis reveals the precursors concerned as representatives of a distinct legumin subfamily, originating from duplication of an ancestral gene prior to or at the beginning of Taxodiaceae diversification.

We have cloned and sequenced two different cDNAs encoding legumins from Japanese red cedar (Cryptomeria japonica, Taxodiaceae). The derived amino acid sequences show between 34% and 55% identity when compared with legumins from angiosperms and from Pinaceae, respectively. The predicted precursors are unusual in that they contain potential glycosylation signals, and we have found the corresponding beta-polypeptides actually to be glycosylated. As most outstanding feature one of the precursors is lacking the Asn-Gly processing site which has been assumed to be highly conserved in legumin gene evolution. Legumin encoding sequences amplified from genomic DNA suggest that these unusual precursors are widespread if not ubiquitous in the Taxodiaceae family. From previous reports on legumin precursors with divergent processing sites, on the proteases involved in legumin precursor processing and from the results presented here it is concluded that the Asn-Gly processing site has been acquired rather than conserved during legumin gene evolution.

The polymerase chain reaction was used to survey gymnosperm legumin genes. Characterization of 46 cloned amplificates, differing in sequence and size (1.2–1.6 kb), revealed the ubiquitous occurrence of legumin genes and their organization in small subfamilies in the 22 species investigated. The 3′ portions of the genes, coding for the legumin β‐polypeptides, show a highly conserved intron/exon structure divergent from those of angiosperms: an additional intron (intron IV) uniformly interrupts the region coding for the C‐terminal part of the β‐polypeptides. Phylogenetic analysis of the respective coding sequences as well as the organization of the Magnolia B14 legumin gene also investigated here both indicate that intron IV is ancestral and was lost during early angiosperm evolution. Taking into account the intronlexon structures from all legumin genes known, our results suggest that legumin genes evolved by subsequent loss of introns, providing also further evidence for a common origin of legumins and vicilins.