Transporter NRT1.5/NPF7.3 suppresses primary root growth under low K+ stress by regulating the degradation of PIN-FORMED2.

BACKGROUND: The availability of potassium is one of the main environmental factors for modifying the plasticity of root architecture. Many potassium channels and transporters are involved in regulating primary root growth in response to low potassium stress. NRT1.5/NPF7.3 transporter is a NO3-/H+ and K+/H+ cotransporter, and participates in NO3- and K+ translocation from the roots to the shoots. However, the underlying mechanism of NRT1.5-regulated primary root growth under low potassium stress is unclear. RESULTS: We show that NRT1.5/NPF7.3 inhibited primary root growth under low potassium conditions by regulating the accumulation of PIN2 protein and auxin levels. Under low potassium conditions, the mutants nrt1.5 and lks2 exhibited longer primary roots, longer meristem regions and elongation zones of primary roots, and more cell activity in the meristem region compared to WT plants, revealing the involvement of NRT1.5 in LK (low potassium)-inhibition primary root growth. In addition, exogenous auxin (IAA), auxin analogue (NAA, 2.4-D) or auxin precursor (IBA) promoted the primary root growth of WT and the complementation line NRT1.5 COM plants. In addition, the application of NPA inhibited the primary root growth of the nrt1.5 and lks2 mutants. Auxin accumulation was higher in the root tip of nrt1.5 plants than in WT plants, indicating that NRT1.5 regulates root growth inhibition by regulating auxin distribution. Furthermore, PIN2 was degraded more quickly in nrt1.5 plants under LK stress. CONCLUSIONS: Our findings reveal that NRT1.5 inhibits primary root growth by modulating the auxin level in the root tip via the degradation of PIN2.

Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa.

Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it is unknown if this mechanism is operative in mushrooms. Herein we report that the native wall extension activity was located exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation growth was restricted also to the 9 mm apical region of the stipes where the elongation growth of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall extension activity represents elongation growth of the stipe. The low concentration of expansin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansin-like protein from snail stomach juice which reconstituted heat-inactivated stipe wall extension without hydrolytic activity. So the previous hypotheses that stipe wall extension was resulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting noncovalent bonding between glucan chains or chitin chains.

Synergism between cucumber alpha-expansin, fungal endoglucanase and pectin lyase.

Several recombinant fungal enzymes (endoglucanase and pectinase) were studied for their interactions with alpha-expansin in cell wall extension and polysaccharide degradation. Both Cel12A and Cel5A were able to hydrolyze cellulose CMC-Na and mixed-linkage beta-glucan. In contrast to Cel5A, Cel12A could also hydrolyze xyloglucan and induce wall extension of cucumber hypocotyls in an in vitro assay. Combining alpha-expansin, even at high concentrations, with Cel12A did not enhance the maximum/final wall extension rate induced by Cel12A alone. These results strongly suggest that modification/degradation of the xyloglucan molecule/network is the key for cell wall extension, and alpha-expansin and Cel12A may share the same acting site in the substrate. Pectinase (Pel1, a pectin lyase) enhanced alpha-expansin-induced wall extension in a concentration-dependent manner, suggesting that the pectin network may normally regulate accessibility of expansin to the xyloglucan-cellulose complex. alpha-Expansin enhanced Cel12A's hydrolytic activity on cellulose CMC-Na but not on xyloglucan and beta-glucan. Expansin did not affect Cel5A's hydrolytic activity. Interestingly, expansin also enhanced Pel1's activity on degrading high esterified pectin. A potential explanation for why expansin could synergistically interact with only certain enzymes on specific polysaccharides is discussed. Additional results also suggested that cell wall swelling may not be a significant event during the action of expansin and hydrolases.

Development of randomly amplified polymorphic DNA-sequence characterized amplified region marker for identification of Apocynum venetum LINN. from A. pictum SCHRENK.

Apocynum venetum LINN. is an important Chinese crude drug, and its sibling species A. pictum SCHRENK is a confusable herb which is similar to it. The purpose of this study is to develop DNA molecular markers to distinguish A. venetum from A. pictum through the combinative technologies of bulked segregate analysis (BSA) and randomly amplified polymorphic DNA (RAPD). Two putative markers B08-407 and B03-1368 specific for A. venetum were identified and sequenced. Based on the sequence information, two pairs of primers were designed and synthesized for sequence characterized amplified region (SCAR) markers. But only one primer pair, B03-1368, produced a clear SCAR band in all samples of A. venetum and not in A. pictum. This SCAR marker was found useful for rapid identification of A. venetum from A. pictum.

Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and EGTA in vitro.

Mature plant cell walls lose their ability to expand and become unresponsive to expansin. This phenomenon is believed to be due to cross-linking of hemicellulose, pectin, or phenolic groups in the wall. By screening various hydrolytic enzymes, we found that pretreatment of nongrowing, heat-inactivated, basal cucumber (Cucumis sativus) hypocotyls with pectin lyase (Pel1) from Aspergillus japonicus could restore reconstituted exogenous expansin-induced extension in mature cell walls in vitro. Recombinant pectate lyase A (PelA) and polygalacturonase (PG) from Aspergillus spp. exhibited similar capacity to Pel1. Pel1, PelA, and PG also enhanced the reconstituted expansin-induced extension of the apical (elongating) segments of cucumber hypocotyls. However, the effective concentrations of PelA and PG for enhancing the reconstituted expansin-induced extension were greater in the apical segments than in the basal segments, whereas Pel1 behaved in the opposite manner. These data are consistent with distribution of more methyl-esterified pectin in cell walls of the apical segments and less esterified pectin in the basal segments. Associated with the degree of esterification of pectin, more calcium was found in cell walls of basal segments compared to apical segments. Pretreatment of the calcium chelator EGTA could also restore mature cell walls' susceptibility to expansin by removing calcium from mature cell walls. Because recombinant pectinases do not hydrolyze other wall polysaccharides, and endoglucanase, xylanase, and protease cannot restore the mature wall's extensibility, we can conclude that the pectin network, especially calcium-pectate bridges, may be the primary factor that determines cucumber hypocotyl mature cell walls' unresponsiveness to expansin.

[The inhibitory effect of Hydrilla verticillata culture water on Microcystic aeruginosa and its mechanism].

This paper studied on the effect and mechanism that the growth of M. aerugonsa was markedly inhibited by the H. verticillata culture water. During treatment, the photosynthetic rate of M. aerugonsa declined, while its respiratory rate and SOD activity increased firstly, then decreased as the treatment went on. Its membrane permeability also increased significantly. TEM photographs showed that the ultrastructure of cell membrane, thylakoid lamella and pith nucleoid of M. aerugonsa were destroyed severely. Inhibitory effects could be observed only when the extracts were extracted by ether. The more extracts from ether, the better inhibitory effect observed. It suggested that the inhibitory effects of H. verticillata on M. aeruginosa were through excreting substances into water. GC/MS analytic result showed that the ether extract mainly consisted of 1,2-benzenedicarboxylic acid diisooctyl ester, dibutyl phthalate, and 1,2-benzenedicarboxylic acid butyl 2-methylpropyl ester.

[Cloning and expression of xyn III from genomic DNA of Trichoderma reesei QM9414 by overlap-PCR].

After the cell enters into its programmed cell death, xylanases from grass plants gradually matured through its N-terminal and C-terminal sequence been cut by acid proteases several times. They could not be expressed by conventional protein expression system. Search the GenBank database, xynIII from a mutant of T. reesei QM9414(ATCC26921)was found. It is similar to grass plants' xylanase in their families and structures. It couldn't express in T. reesei QM9414, but its gene exist in genomic DNA as one copy. Through overlap-PCR method, 4 exons of xynIII were cloned, sequenced, spliced, and the whole cDNA of mature xynIII was acquired. The cDNA was inserted into pETBlue-2 vector and transformed into E. coli DE3 pLacI cell. Xyn III could be expressed in the transformed cell under the conditions of 37 degrees C, 1 mmol/L IPTG induced for 3h. Low temperature (15 degrees C), long time(64h) induction(0.2 mmol/L IPTG) could enhance xynIII activity.