Kenneth R. Markham

Flavonoid biosynthesis in flower petals of five lines of lisianthus (Eustoma grandiflorum Grise.)

Abstract The biosynthesis of flavonoid pigments during petal development was investigated in five lines of lisianthus ( Eustoma grandiflorum Grise.), with purple, pink, mauve or white flowers (W42 and W84). Flavonols were found at high levels at all stages of petal development, but peaked just prior to flower opening. In the cyanic lines, anthocyanins were first detected in opening flowers, increasing in amounts thereafter. The polymerase chain reaction was used to generate cDNAs from lisianthus petal RNA for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and dihydroflavonol 4-reductase (DFR), and expression of their corresponding mRNAs during petal development investigated. In the cyanic lines, PAL, CHS and CHI transcripts were present throughout petal development, with peak expression in young, acyanic buds. DFR expression was low in young buds and peaked immediately preceding anthocyanin synthesis. Thus, the activity of the biosynthetic genes correlated closely with the flavonoid content of the petals, with the synthesis of flavonols being temporally separated from that of anthocyanins. Transcripts for all four genes examined were detected in both white-flowered lines, although line W42 had only low levels for DFR. Neither white-flowered line could synthesize anthocyanins when petal tissue was fed with leucopelargonidin and leucocyanidin (fed to W42 only), products of the DFR enzyme. Thus, line W42 may lack activity for a regulatory factor that controls the expression of the genes of the later part of the flavonoid biosynthetic pathway, while W84 may contain a block in the conversion of leucoanthocyanidin to anthocyanin.

Floral flavonoids and the potential for pelargonidin biosynthesis in commercial chrysanthemum cultivars

Abstract The results of a survey of flavonoids in the ray florets of 38 commercial chrysanthemum (Dendranthema grandiflora) cultivars and one D. grandiflora cross are reported. The major flavonoids found were apigenin, acacetin, luteolin, diosmetin and eriodictyol, which occurred variously as 7-O-glucosides, 7-O-malonylglucosides, 7-O- glucuronides and 7-O-rutinosides. A number of minor flavonoids were also identified. Many of these compounds have not been observed before in D. grandiflora and one, acacetin 7-O-β-(6″-O-malonylglucoside), is reported for the first time as a natural product. An investigation of anthocyanin biosynthesis in chrysanthemum cultivars, which normally produce cyanidin glycosides, was also conducted. Flavonoid 3′-hydroxylase activity was demonstrated to be responsible for the lack of pelargonidin-based pigments in the flowers.

Expression of an Antirrhinum majus UDP-glucose : flavonoid-3-O-glucosyltransferase transgene alters flavonoid glycosylation and acylation in lisianthus (Eustoma grandiflorum Grise. )

Abstract A binary vector containing an Antirrhinum majus UDP-glucose:flavonoid-3- O -glucosyltransferase (UFGT) cDNA under the control of the cauliflower mosaic virus 35S promoter was used to transform lisianthus ( Eustoma grandiflorum Grise.). Of four independent transgenic lines recovered, one produced high levels of the UFGT transcript and synthesized 3- O -glucosylated anthocyanins novel to lisianthus, as well as enhanced levels of 3- O -glucosylated flavonols. The novel 3- O -glucosylated anthocyanins accounted for approximately 30% of the total anthocyanins in the petals. The level of 3- O -glucosylated flavonols increased by approximately 4.8 x , representing a change from the production of 3- O -galactosylated flavonols to 3- O -glucosylated flavonols of approximately 32%. In addition, the increase in glucosylation of the flavonols resulted in a corresponding decrease in acylation. Modifications at other positions of the flavonoid nuclei were unaffected. The results indicate that in lisianthus there is a high specificity shown by some flavonoid modifying enzymes, such as that for flavonol acylation, while other enzymes, such as those involved in modifications at the C-5 of the anthocyanin, are more flexible in their choice of substrate. The results also provide information on the role of flavonoid glycosylation and acylation in copigmentation in lisianthus.

Antisense flavonol synthase alters copigmentation and flower color in lisianthus

In order to generate new flower colors in lisianthus, a flavonolsynthase (FLS) coding sequence was isolated from lisianthus (Eustomagrandiflorum Grise.) using a petunia homologue (pCGP481) as a probe.The endogenous FLS mRNA transcript occurs early in petal development,concomitant with accumulation of flavonols in bud tissue, and ceases at onsetofanthocyanin pigment production. Southern DNA analysis indicated FLS as a memberof a multigene family in lisianthus. Transgenic plants of a purple floweredlisianthus line expressing antisense FLS under the control of the CaMV35Spromoter produced flowers more red in color (magenta) than the originaluntransformed plant. The transgenic plants also showed novel red pigmentation(cyanidin) in early stage buds as well as deeply colored pistils, andaccumulated dihydroflavonols at the expense of flavonols. Further, analysis ofthe progeny of crosses between the primary transformants and a deeply pigmentedpurple variety (Wakamurasagi) showed this novel phenotype to be stablyinherited. Field trial assessment of antisense FLS plants indicated variationinthe petal and early bud phenotype although variation in the transgenic flowercolor was within the range normally seen in commercial cultivars grown undercommercial conditions.

A taxonomic revision of the Hebe parviflora complex (Scrophulariaceae), based on morphology and flavonoid chemistry

Abstract Two entities have long been recognised at the rank of either species or variety in the New Zealand endemic Hebe parviflora complex but, because one of the critical type specimens had not previously been examined by New Zealand botsnists, there has been uncertainty regarding the correct application Of names. The two entities Can be distinguished on characters of habit, leaves, flowers, leaf flavonoids, and chromosome number, and are accepted here at species rank. H. parvlflora is the correct name for the tetraploid entity previously known as H. parvrflora var. arborea, and H. stenophylla is the diploid entity previously known as H. parviflora var. angustifolia (the autonym var. parviflora has not been used in any recent treatments). H. stenophylla has several distinct geographic races and new names are provided at varietal rank for two of these: var. hesperia occurs in limestone areas near the north‐west coast of the South Island, between Cape Farewell and the Heaphy River; var. oliveri occurs on exposed bluffs on Stephens Island in Cook Strait. Circumscription of these varieties leaves var. stenophylla comprising a range of morphological forms whose relationships require further elucidation. A comparative study of leaf flavonoids identifies flavonoid characters that clearly distinguish H. parviflora and H. stenophylla, and the profiles of these species are compared with those of the related species H. strictissima and H. traversii. Historical factors affecting the distribution of H. parviflora and H. stenophylla are discussed, and descriptions, distribution maps, and illustrations of diagnostic morphological characters are provided for all taxa.

Description and flavonoid chemistry of Hebe calcicola (Scrophulariaceae), a new species from north-west Nelson, New Zealand

Abstract Hebe calcicola is a new species described from outcropping marble in north‐west Nelson, New Zealand. It is a shrub with glossy leaves to 45 mm long, no sinus in the leaf bud, unbranched and lateral inflorescences, pedicillate flowers, white corollas that are papillate within with tubes shorter than surrounding calyces, and pubescent ovaries and fruit. It is placed in series Occlusae, and is morphologically and geographically distinguished from other members of that group. A comparative study of leaf flavonoids has identified flavonoid characters that Clearly distinguish H. calcicola from the morphologically similar species H. rakaiensis, H. subalpina, H. truncatula, H. traversii, and H. strictissima, and supports morphological evidence for the existence of naturally occurring hybrids between H. calcicola and both H. albicans and H. salicifolia.

Geographic variation in morphology and flavonoid chemistry in Hebe pubescens and H. bollonsii (Scrophulariaceae), including a new infraspecific classification for H. pubescens

Abstract Geographic variation in morphology, leaf anatomy, and flavonoid chemistry is appraised in Hebe pubescens, H. bollonsii, and allied plants from islands of outer Hauraki Gulf, North Island, New Zealand. The circumscription of H. pubescens is modified to include plants from Great Barrier, Little Barrier, and the Mokohinau Islands, as well as the Coromandel Peninsula and immediately surrounding islands. A new infraspecific classification is proposed for H. pubescens, wherein three subspecies are recognised. Subspecies pubescens occurs on the Coromandel Peninsula and immediately surrounding islands, and also at Papanui Point on the western coast of the Firth of Thames. Subspecies rehuarum occurs on Great Barrier Island. Subspecies sejuncta occurs primarily on the Mokohinau Islands and Little Barrier Island, with a single plant also known on Great Barrier Island. Populations of H. pubescens probably form a cline of variation, and some forms of subsp. sejuncta share morphological features with H. bollonsii, which is here considered restricted to the Poor Knights Islands, the Hen and Chickens Islands, and nearby areas of the Northland coast. Possible relationships of H. pubescens and H. bollonsii are discussed. A key to the taxa of Hebe pubescens is provided.

Descriptions and flavonoid chemistry of new taxa in Hebe sect. subdistichae(Scrophulariaceae)

Abstract This paper is a further contribution toward a revision of the species‐level taxonomy of Hebe in New Zealand. Data on morphology, flavonoid chemistry, and chromosome numbers are used in the recognition of new taxa of section Subdistichae Heads. New taxa described herein are: H. societatis, known only from Mt Murchison, Braeburn Range, South Island; H. scopulorum, which occurs in the Awaroa valley, south‐east of Kawhia, North Island; H. rigidula var. sulcata, which occurs on DUrville Island and nearby areas of northern South Island; H. crenulata, which occurs in mountains of western and central areas of the northern South Island, between the Douglas Range in the north‐west and the Poplars Range in the southeast; and H. cryptomorpha, which occurs primarily on mountains of the Wairau River catchment, South Island. The latter two species cannot always be separated on morphological grounds, and the decision to recognise both at species rank is based on differences in flavonoid chemistry and chromosome number. Distribution maps and illustrations of morphological features are provided for all new taxa.

Variation in morphology and flavonoid chemistry in Hebe pimeleoides (Scrophulariaceae), including a revised subspecific classification

Abstract Variation in morphology and flavonoid chemistry is appraised in populations of Hebe pimeleoides. An infraspecific classification is proposed for H. pimeleoides, wherein two subspecies are recognised. Subspecies pimeleoides occurs from south‐east Nelson to central Otago, and is a subshrub of open places near lakes and streams, and of grassy slopes. Subspecies faucicola occurs in central Otago, and is a small, erect shrub growing on rocky outcrops in river valleys. There is some morphological overlap between the two subspecies, and two chromosome numbers (n = 20, n = 40) have been recorded in each. Subspecies faucicola is recognised on the basis of its distinctive habit and habitat, as well as correlated trends in other morphological characters (particularly flower colour) and leaf flavonoid content. A key to the subspecies of Hebe pimeleoides is provided.

A taxonomic revision of Hebe informal group “Connatae” (Plantaginaceae∗), based on morphology and flavonoid chemistry

Abstract A revised species‐level classification is presented for Hebe informal group “Connatae”, based on a study of morphology and leaf flavonoids. Accepted species are: Hebe macrocalyx (including var. macrocalyx and var. humilis), H. haastii, H. epacridea, H. ramosissima, H. petriei, H. murrellii, and H. benthamii. The limits of H. macrocalyx, H. haastii, H. epacridea, and H. ramosissima have historically been the subject of confusion and/or debate, and detailed accounts of these taxa are presented. In particular, H. epacridea is morphologically more variable and more widespread than previous treatments suggest, and it has sometimes been confused with the more geographically restricted H. haastii. The name H. macrocalyx var. humilis (H. haastii var. humilis in some accounts) has sometimes been misapplied. It is used here for wild populations known only from Nelson and Marlborough, although this remains at odds with stated source locations of the cultivated specimens cited in the protologue (Otago and Southland). A key to all taxa of “Connatae” is provided.