Ethylene and IAA interactions in the inhibition of photoperiodic flower induction of <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It has been shown that both IAA and ethylene application inhibit flower induction in the short-day plant Pharbitis nil. However application of IAA has elevated ethylene production in this plant, as well. Strong enhancement of ethylene production is also correlated with the night-break effect, which completely inhibits flowering. In order to determine what the role of IAA and ethylene is in the photoperiodic flower induction in Pharbitis nil, we measured changes in their levels during inductive and non-inductive photoperiods, and the effects of ethylene biosynthesis and action inhibitors on inhibition of flowering by IAA. Our results have shown that the inhibitory effect of IAA on Pharbitis nil flowering is not physiological but is connected with its effect on ethylene biosynthesis.     

The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm

When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction.     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

ISOLATION AND CHARACTERIZATION OF PHASE-SPECIFIC COMPLEMENTARY DNAs FROM SPOROPHYTES AND GAMETOPHYTES OF PORPHYRA PURPUREA (RHODOPHYTA) USING SUBTRACTED COMPLEMENTARY DNA LIBRARIES1

The red alga Porphyra purpurea (Roth) C. Agardh has a life cycle that alternates between shell-boring, filamentous sporophytes and free-living, foliose gametophytes. The significant morphological differences between these two phases suggest that many genes should be developmentally regulated and expressed in a phase-specific manner. In this study, we prepared and screened subtracted complementary DNA (cDNA) libraries specific for the sporophyte and gametophyte of P. purpurea. This involved the construction of cDNA libraries from each phase, followed by the removal of common clones through subtractive hybridization. Sampling of the subtracted libraries indicated that 8–10% of the recombinant colonies in each library were specific for the appropriate phase. Of 20 putative phase-specific cDNAs selected from each subtracted library, eight unique clones were obtained for the sporophyte and seven for the gametophyte. After confirming their phase-specificities by hybridization to gametophyte and sporophyte messenger RNA, these 15 phase-specific cDNAs were sequenced, and the deduced amino acid sequences were used to search protein databanks. Two proteins encoded by the sporophyte-specific cDNAs and two by the gametophyte-specific cDNAs were identified by their similarity to databank entries.     

Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species. This revised version was published online in June 2006 with corrections to the Cover Date.     

大孔吸附树脂对紫锥菊提取物中菊苣酸分离纯化的研究

采用大孔吸附树脂分离纯化紫锥菊提取物中免疫活性成分菊苣酸,7％(v／v)的甲醇-水溶液洗脱,HPLC法对产品中菊苣酸含量进行检测分析。该法能把紫锥菊提取物中菊苣酸含量(4％)提高9倍左右,回收率达到95％以上,且操作简单,可用于菊苣酸的分离纯化。     

Axillary bud flowering after apical decapitation in Pharbitis in relation to photoinduction

The flowering response of axillary buds of seedlings of Pharbitis nil Choisy, cv. Violet, was examined in relation to the timing of apical bud removal (plumule including the first leaf or second leaf) before or after a flower-inductive 16-h dark period. When the apical bud was removed well before the dark period, flower buds formed on the axillary shoots that subsequently developed, but when removed just before, or after, the dark period, different results were observed depending on the timing of the apical bud removal and plant age. In the case of 8-day-old seedlings, fewer flower buds formed on the axillary shoots developing from the cotyledonary node when plumules were removed 20 to 0 h before the dark period. When the apical bud was removed after the dark period, no flower buds formed. Using 14-day-old seedlings a similar reduction of flowering response was observed on the axillary shoots developing from the first leaf node when the apical bud was removed just after the dark period. To further elucidate the relationship between apical dominance and flowering, kinetin or IAA was applied to axillary buds or the cut site where the apical bud was located. Both chemicals influenced flowering, probably by modulating apical dominance which normally forces axillary buds to be dormant.     

象牙参种子的解剖学和组织化学研究

象牙参种子解剖学和组织化学的研究结果表明, 种子包括假种皮、种皮、外胚乳、内胚乳和胚。假种皮没有完全包被种子, 由约4~5 层薄壁细胞构成。种皮可以分为外种皮、中种皮和内种皮。外种皮由1 层表皮细胞构成, 细胞壁明显增厚;中种皮包括下皮层、半透明细胞层和3~4层细胞的色素层, 下皮层和色素层细胞均充满红棕色色素;内种皮由1 层体积小、壁局部增厚的砖形薄壁细胞构成。种子在珠孔端分化出珠孔领、孔盖和种阜状结构, 珠孔领为同形型, 孔盖不具石细胞硬层。合点区内种皮出现缺口, 缺口间充满合点区色素细胞, 其整体轮廓成新月形。外胚乳可分为厚区与薄区两部分, 外胚乳细胞壁平直, 细胞内充满淀粉。内胚乳细胞主要含蛋白质, 也有少量脂类物质, 细胞界限不清楚。胚棒状, 两端略膨大, 含大量脂类物质, 也含蛋白质和多糖。     

Allene oxide cyclase is essential for theobroxide-induced jasmonic acid biosynthesis in Pharbitis nil

Theobroxide, a natural product, strongly stimulates the biosynthesis of jasmonic acid (JA) in Pharbitis nil. In this study, we investigated the accumulation of protein by the immunoblot analysis of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC), key enzymes in JA biosynthesis, and how the endogenous levels of JA in P. nil are affected by theobroxide. The effect of JA on the accumulations of these proteins was monitored simultaneously. The results show that theobroxide treatment led to a high level accumulation of JA, which is due to high accumulations of LOX, AOS, and AOC proteins induced by theobroxide treatment both under short day (SD) and long day (LD) conditions. However, under SD conditions AOS and AOC proteins are not enhanced by JA treatment. Kinetic analysis of protein levels shows that a biphasic activation of AOC protein by theobroxide is displayed and the first activation of AOC protein together with elevated JA levels is observed within 30min after treatment. Meanwhile, AOS and LOX proteins are activated by theobroxide later than AOC protein, suggesting that AOC plays an essential role in the initial JA formation induced by theobroxide. Since theobroxide-increased JA levels also show a biphasic manner similar to AOC activation and AOS, LOX proteins are activated later than AOC, and thus we propose a positive JA feedback regulation. Interestingly, AOS protein, which is also the enzyme for the biosynthesis of 9,10-ketol-octadecadienoic acid (KODA, a flowering inducing factor), accumulates markedly due to the simultaneous involvement of theobroxide and SD conditions, suggesting that AOS probably plays a role in flower bud formation in P. nil.     

紫果猕猴桃幼胚愈伤组织诱导及植株再生

以紫果猕猴桃(Actinidia arguta var．purpurea)幼胚为外植体,诱导愈伤组织并进行植株再生。结果表明：不同的培养基和不同的培养条件对幼胚愈伤组织的诱导率及分化率不同;0．2mg／L ZT与0．5mg／L GA。配合使用有利于促进愈伤组织的诱导;7％蔗糖、600mg／L CH与400mg／L Gln都有利于促进愈伤组织的形成;在添加0．5mg／L 6-BA、0．05mg／L NAA与0．5mg／L GA3的MS培养基中植株的再生率达93．3％。