Arabidopsis RNA Isolation Plants often present a challenge to researchers wanting to isolate nucleic acids for subsequent analyses as a result of the high amounts of polysaccharides and other contaminating metabolites present in plant tissues. Suzuki et al. (p. 542) describe an isolation method that produces RNA free of contamination by protein or polysaccharides or degradation, suitable for reverse transcription PCR (RT-PCR). The authors extracted RNA from Arabidopsis siliques, seeds, flower buds, leaves, roots, and stems with no decrease in the quality or quantity of RNA. Their method was both time- and cost-effective over more conventional methods and is simple enough to be routinely applied to other species.

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The gene encoding ,-amylase was cloned from Bacillus polymyxa 72 into Escherichia coli HB101 by inserting HindIll-generated DNA fragments into the Hindm site of pBR322. The 4.8-kilobase insert was shown to direct the synthesis of ,-amylase. A 1.8-kilobase AccI-AccI fragment of the donor strain DNA was sufficient for the I8-amylase synthesis. Homologous DNA was foun4 by Southern blot analysis to be present only in B. polymyxa 72 and not in other bacteria such as E. coli or B. subtilis. B. polymyxa, as well as E. coli harboring the cloned DNA, was found to produce enzymatically active fragments of (-amylases (70,000, 56,000, or

Eucalyptus is a diverse genus of flowering trees with more than 700 genotypic species which are mostly native to Australia. We selected 19 wild provenances of Eucalyptus camaldulensis grown in Australia, compared their growth rate and drought tolerance and determined the protein levels of plasma membrane aquaporins (PIPs). There was a positive relationship between the drought tolerance and PIP content. PIPs are divided into two subgroups, PIP1 and PIP2. Most members of the PIP2 subgroup, but not PIP1 subgroup, exhibit water channel activity. We introduced two radish (Raphanus sativus L.) PIPs, RsPIP1;1 and RsPIP2;1, into a hybrid clone of Eucalyptus grandis and Eucalyptus urophylla to examine the effect of their overexpression. Expression of these genes was confirmed by real-time polymerase chain reaction (PCR) and the protein accumulation of RsPIP2;1 by immunoblotting. Drought tolerance was not enhanced in transgenic lines of either gene. However, one transgenic line expressing RsPIP2;1 showed high photosynthesis activity and growth rate under normal growth conditions. For RsPIP1;1-transformed lines, the RsPIP1;1 protein did not accumulate, and the abundance of endogenous PIP1 and PIP2 was decreased. The endogenous PIP1 and PIP2 genes were suppressed in these lines. Therefore, the decreased levels of PIP1 and PIP2 protein may be due to co-suppression of the PIP genes and/or high turnover of PIP proteins. RsPIP1;1-expressing lines gave low values of photosynthesis and growth compared with the control. These results suggest that down-regulation of PIP1 and PIP2 causes serious damage and that up-regulation of PIP2 improves the photosynthetic activity and growth of Eucalyptus trees.

The dynamics of ribulose 1·5-bisphosphate carboxylase/ oxygenase (Rubisco) content and turnover during leaf development are not well understood in woody plants. Rubisco synthesis, N influx and the mRNA levels of Rubisco-encoding genes were determined as a function of leaf position in 4.5-month-old Eucalyptus globulus seedlings. Rubisco concentration was slightly higher in the top leaves as leaf expansion progressed and was almost maximal in the uppermost fully expanded leaves. Rubisco concentration remained almost constant in the fully expanded leaves at the top and middle positions and then became slightly low at the lowest positions. Rubisco synthesis was active only in the top leaves. These results suggest that Rubisco turnover rate is low in the middle leaves, leading to the maintenance of Rubisco contents, and that Rubisco degradation primarily occurs in the lowest leaves. Changes in the RBCS and rbcL mRNA levels were roughly parallel with Rubisco synthesis, but N influx was more closely correlated with Rubisco synthesis. These results suggest that N influx rather than the transcript abundance of Rubisco-encoding genes is of primary importance in regulating the rate of Rubisco synthesis. Additionally, expression of RBCS multigene family in E. globulus leaves was discussed.

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Many plant species excrete organic acids into the rhizosphere in response to aluminum stress to protect sensitive cells from aluminum rhizotoxicity. When the roots of Eucalyptus camaldulensis, a major source of pulp production, were incubated in aluminum-toxic medium, citrate released into the solution increased as a function of time. Citrate excretion was inducible by aluminum, but not by copper or sodium chloride stresses. This indicated that citrate is the major responsive organic acid released from the roots of this plant species to protect the root tips from aluminum damage. Four genes highly homologs to known citrate-transporting multidrugs and toxic compounds exclusion proteins, named EcMATE1-4, were isolated using polymerase chain reaction-based cloning techniques. Their predicted proteins included 12 membrane spanning domains, a common structural feature of citrate-transporting MATE proteins, and consisted of 502-579 amino acids with >60 % homology to orthologous genes in other plant species. One of the homologs, designated EcMATE1, was expressed in the roots more abundantly than in the shoots and in response to both Al and low pH stresses. Ectopic expression of EcMATE1 and 3 in tobacco hairy roots enhanced Al-responsive citrate excretion. Pharmacological characterization indicated that Al-responsive citrate excretion involved a protein phosphorylation/dephosphorylation process. These results indicate that citrate excretion through citrate-transporting multidrugs and toxic compounds exclusion proteins is one of the important aluminum-tolerance mechanisms in Eucalyptus camaldulensis.

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