Isolation and characterization of the orchid cytokinin oxidase DSCKX1 promoter

The orchid DSCKX1 is a new member of the cytokinin oxidase gene family, which catalyses the degradation of cytokinins bearing unsaturated isoprenoid side chains. A 3.7 kb fragment upstream of the DSCKX1 coding region was isolated, sequenced and characterized by deletion analysis of DSCKX1::beta-glucuronidase gene fusions using transient orchid and stable Arabidopsis transformation systems. Functional analysis of 5' deletions defined the 5'-upstream region that directs the expression in distinct tissues. Regulatory elements affecting the cytokinin induction of the DSCKX1 gene have also been delineated     

两种穗型冬小麦籽粒淀粉积累动态及其有关酶活性变化

花后25d内,大穗型品种豫麦66籽粒中淀粉积累比多穗型品种豫麦49慢,但花后25d后情况则相反。2个品种籽粒中淀粉积累速率的变化均呈单峰曲线,豫麦49峰值出现在花后15～20d,而豫麦66峰值则出现在花后20～25d。灌浆期豫麦66和豫麦49籽粒中蔗糖合成酶(SS)活性变化呈单峰曲线,峰值分别出现在花后20d和15d,整个灌浆期内豫麦66籽粒中SS活性高于豫麦49;2个品种籽粒中腺苷二磷酸葡萄糖焦磷酸化酶(AGPP)和淀粉分支酶(SBE)活性变化均呈单峰曲线,峰值出现在花后20d,而可溶性淀粉合成酶(SSS)活性变化则呈双峰曲线,峰值分别出现在花后10d和20d,且第二个峰值显著高于第一个。相关分析表明,SS、AGPP、SSS和SBE是影响小麦籽粒淀粉积累的关键酶。     

Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs

Cytokinin oxidases (CKOs) play a major role in the regulation of hormone levels in plants by irreversibly degrading cytokinins. Two new cDNAs from maize (CKO2 and CKO3) were cloned and CKO activity of a recombinant CKO3 enzyme was demonstrated. CKO2 and CKO3 encode flavoproteins with 93% identity among each other compared with 45% identity with CKO1. The respective genes were mapped to BIN 3.05/06 and BIN 8.06 which belong to duplicated regions of the maize genome. For a better understanding of the role of CKO2 and CKO3 in maize development, their expression profiles were analysed in different organs and during kernel development via semi-quantitative RT-PCR. Different spatial and temporal expression patterns were observed for the two genes, as well as for CKO1 and two additional genes CKO4 and CKO5. CKO2 to CKO5 genes were mainly expressed in vegetative tissues, with unique expression patterns. CKO1 was most strongly expressed in the kernel. All five genes were expressed at early stages of kernel development, a period when a peak in cytokinin levels and a high cell division rate in the endosperm have been described. However, each gene had its own expression profile with a major difference concerning the onset of expression.     

Isolation and characterization of three polyamine oxidase genes from Zea mays

Isolation and sequencing of three genes, MPAO1, MPAO2 and MPAO3, coding for polyamine oxidase (PAO) from maize (Zea mays) are reported here. Gene organization is extremely conserved among these copies, being composed of eight exons and seven introns. Furthermore, these genes encode for a protein of an almost identical amino acid sequence. These data suggest that the three MPAO copies have been derived from gene duplication of a common ancestor gene. Long inverted repeat sequences, also present in other maize genes, have been found within the second intron. Promoter sequences of MPAO1 and MPAO2 genes have been analysed for putative cis-acting elements. According to genomic Southern blot analysis, the MPAO gene family in maize and other monocots is represented by a small number of copies. Northern and western blot analysis have revealed a tissue-specific accumulation of both MPAO mRNA and protein.     

Isolation and characterization of two wound-regulated tomato extensin genes

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library byin situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro₄-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro₄-Tyr₃-Lys-Ser-Pro₄-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro₄ repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro₄-Ser-Pro-Ser-Pro₄-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.     

Wild-type alleles of Rht-B1 and Rht-D1 as independent determinants of thousand-grain weight and kernel number per spike in wheat

The utilization of dwarfing genes Rht-B1b and Rht-D1b in wheat significantly increased grain yield and contributed to the “green revolution”. However, the benefit of Rht-B1b and Rht-D1b in drought environments has been debated. Although quantitative trait loci (QTL) for kernel number per spike (KN) and thousand-grain weight (TGW) have been found to be associated with Rht-B1 and Rht-D1, the confounding effect of environmental variation has made a direct association difficult to find. In this study, we used a doubled haploid population (225 lines) of Westonia × Kauz, in which both Rht-B1b (Kauz) and Rht-D1b (Westonia) segregated. The purpose of the study was to determine the interaction of Rht-B1 and Rht-D1 with grain yield components, namely KN and TGW, and to investigate genotype-by-environment interactions in glasshouse and field trials conducted in 2010 and 2011 in Western Australia. A genetic map of 1,156 loci was constructed using 195 microsatellite markers, two gene-based markers for Rht-B1 and Rht-D1, and 959 single nucleotide polymorphisms. The major QTL for TGW and KN were strongly linked to Rht-B1 and Rht-D1 loci and the positive effects were associated with the wild-type alleles, Rht-B1a and Rht-D1a. The major QTL of TGW were on chromosome 2D and 4B. The significant genetic effects (14.6–22.9 %) of TGW indicated that marker-assisted selection for TGW is possible, and markers gwm192a (206 bp) or gwm192b (236 bp) can be used as indicators of high TGW. For KN, one major QTL was detected on chromosome 4D in the analysis across three environments. The association of the wild-type alleles Rht-B1a and Rht-D1a in drought environments is discussed.     

Differential regulation of cytokinin oxidase genes and cytokinin-induced auxin biosynthesis by cellular cytokinin level in transgenic poplars

Key message

The present work with transgenic poplar lines producing varying levels of trans -zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin.

Abstract

Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4 appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis.     

Molecular and biochemical characterization of a cytokinin oxidase from maize

It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described.     

Isolation and characterization of two knotted-like homeobox genes from tomato

Homeobox genes are known to play a role in developmental regulation. The knotted-like homeobox (knox) genes fall into two classes. The class I knox genes like kn1, stm1, and knat1 are involved in maintaining meristem identity in cells. The function of class II knox genes is at yet undetermined. We have characterized two knox genes from tomato. LeT6 and LeT12 map to distinct chromosome locations that are different from the location for a recently cloned knox gene from tomato, tkn1, confirming that plant homeobox genes are not clustered on chromosomes. These genes have a distinct expression pattern. Unlike other class I kn1-like genes, LeT6 is expressed in developing lateral organs and developing ovaries in flowers. LeT12 is more ubiquitously expressed in the mature plant. RNA in situ localization data suggest that both these genes may have a role to play in formative events in ovule and embryo morphogenesis.     

Molecular cloning and expression patterns of three putative functional aldehyde oxidase genes and isolation of two aldehyde oxidase pseudogenes in tomato

The final steps in the biosynthesis of the plant hormones abscisic acid (ABA) and indole-3-acetic acid (IAA) have been shown to be catalyzed by aldehyde oxidases (AO). We have cloned three putative functional AO genes (TAO1, TAO2 and TAO3) and two putative AO pseudogenes (TAO4 and TAO5) in tomato. The TAO1 cDNA described here includes the correct amino terminus of the encoded TAO1 protein and is different at the 5'-end from the TAO1 sequence in GenBank (accession number U82558). Northern analysis shows that TAO1 is expressed mainly in vegetative tissues and TAO2 is expressed in both vegetative and reproductive tissues. TAO3 expression was not detectable by Northern hybridization. These results suggest that each AO may play different roles in the regulation of tomato growth and development.     

Isolation and characterization of chicken DNA homologous to the two putative oncogenes of avian erythroblastosis virus

The genome of avian erythroblastosis virus contains two independently expressed genetic loci (v-erbA and v-erbB) whose activities are probably responsible for oncogenesis by the virus. Both loci are closely related to nucleotide sequences found in the DNA and RNA of chickens and other vertebrates. We have isolated and characterized chicken DNA homologous to v-erbA and v-erbB. The two viral genes are represented by separate domains within chicken DNA (c-erbA and c-erbB), which are separated by a minimum of 12 kilobases (kb) of DNA and may not be linked at all. The nucleotide sequences shared by the viral and cellular erb loci are colinear, but the cellular loci are interrupted by multiple intervening sequences of various lengths. Polyribosomes prepared from normal chicken embryos contain two polyadenylated RNAs transcribed from c-erbA and two transcribed from c-erbB. The evident coding regions of these RNAs represent an unusually small fraction of the lengths of the RNAs, as if the 3′ untranslated domains of the RNAs might be exceptionally large (3–11 kb). These findings indicate that the c-erb loci are normal vertebrate genes rather than genes of cryptic endogenous retroviruses, and that they may have a role in the metabolism of normal cells. It appears that the viral erb genes, like most other retrovirus oncogenes, have been copied from cellular genes. In the viral genome, the two genes are devoid of introns, but they remain independently expressed loci, and they remain colinear with the coding domains of their cellular progenitors.     

两个小麦磷转运蛋白基因的分离、功能鉴定和表达研究

磷是能量代谢、核酸以及许多生物膜合成的重要底物。在光合作用、呼吸作用等过程中发挥了重要作用。中国大多数小麦产区的土壤存在着缺磷的问题。磷饥饿给小麦生产造成了很大损失。培育耐低磷小麦是解决这一问题的一个重要途径。在磷饥饿的过程中,哪些基因的表达发生了变化．它们是如何变化的,弄清楚这些问题对于培育转基因耐低磷小麦具有重要的意义。磷转运蛋白基因在植物吸收磷的过程中发挥着重要作用。利用RT—PCR的方法,我们从普通小麦“小偃54”中分离了两个磷转运蛋白基因TaPT8和TaPHT2;1。通过与酵母突变体互补分析表明这两个基因都能够与磷吸收功能存在缺陷的酵母突变体实现功能互补,在低磷条件下有促进酵母突变体吸收磷的作用。进一步分析表明TaPT8属于Pht1家族。TaPHT2;1属于Pht2家族。运用RQRT—PCR的方法进行分析后发现TaPT8在根中表达,受磷饥饿的诱导;TaPHT2;1主要在绿色组织中表达,受磷饥饿的抑制,受光的诱导。TaPT8可能主要参与了小麦的根从土壤中吸收磷的过程。TaPHT2;1可能在磷从细胞质向叶绿体内转运的过程中发挥了重要作用。     

Effects of crop thinning and reduced grain numbers per ear on grain size in two winter wheat varieties given different amounts of nitrogen

To study the importance for final grain size in wheat (Triticum aestivum, L.) of assimilate supply and the storage capacity of the grain, two field experiments were done. In 1976 nitrogen was applied in the range from none to 180 kg ha^-1, part of the crop was thinned, and the top halves of some ears of the short variety Hobbit and of the tall variety Maris Huntsman were removed soon after anthesis. In 1977 ears of Maris Huntsman were halved 5 days after anthesis or at 30 days after anthesis when grain volume was maximum. Thinning the crop from 360 to 180 ear-bearing shoots m^-2 30 days before anthesis increased the number of grains per ear, except in the absence of nitrogen fertiliser, but did not increase grain size, grain dry weight per ear or total dry weight per culm. Removing the upper half of ears of Hobbit 5 days after anthesis increased dry weight per grain, but when this treatment was applied to Maris Huntsman either 5 days after anthesis in 1976 and 1977, or when grain volume was maximal in 1977, the grains failed to increase in dry weight. Non-grain dry weight of both varieties was increased by halving the ear. In both varieties the maximum volume of grains in halved ears was larger than in intact ears. Grain dry weight increased relatively less than volume after halving the ear of Hobbit, and the decrease in volume up to maturity was greater in halved than intact ears of both varieties. The larger grain volume in halved ears of Maris Huntsman in 1977 was associated with more endosperm cells.     

Molecular characterization and functional analysis of elite genes in wheat and its related species

The tribe Triticeae includes major cereal crops (bread wheat, durum wheat, triticale, barley and rye), as well as abundant forage and lawn grasses. Wheat and its wild related species possess numerous favourable genes for yield improvement, grain quality enhancement, biotic and abiotic stress resistance, and constitute a giant gene pool for wheat improvement. In recent years, significant progress on molecular characterization and functional analysis of elite genes in wheat and its related species have been achieved. In this paper, we review the cloned functional genes correlated with grain quality, biotic and abiotic stress resistance, photosystem and nutrition utilization in wheat and its related species.