Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.     

大豆球蛋白G1启动子的克隆及植物表达载体构建

从大豆冀nf37和冀豆15中克隆了大豆球蛋白G1基因的启动子片段。序列分析表明,两种启动子片段均为688bp,与GenBank现有的3种启动子序列(四川大豆(DQ250808)、南农87-c38(AY649096)和Dare(X15121))间的同源性在96.4%～99.6%之间。其中来自冀nf37的启动子片段除Legumin盒上有一个碱基差异外,其它元件与DQ250808完全相同,据此推测该启动子片段具有种子特异性启动子活性。将其与已有γ-生育酚甲基转移酶基因连接,构建了种子特异性表达载体pBG1TMT,为通过代谢工程手段调控油料作物种子维生素E组成、提高其营养品质奠定了基础。     

大豆基因家族的结构和表达分析

KNOX基因家族编码同源异型盒蛋白,在植物生长发育过程中起重要调控作用。利用生物信息学手段在全基因组水平上对大豆（Glycine Max）KNOX）（家族基因进行鉴定和分类,并分析其基因结构、蛋白同源结构域特征以及基因表达方式。研究结果表明：大豆中的27个GmKNOX基因可以分为GmKNOXl和GmKNOXll两个亚类,其中GmKNOXl类可分为3个主要的进化支,GmKNOXll类分为2个主要的进化支：26个GmKNOX基因不均匀地分布在16条染色体上,GmKNOX27尚无法定位。不同组织表达谱的分析表明：GmKNOXl类基因表达部位比较集中,以茎顶端分生组织中表达量最高：而GmKNOXll类基因的表达特异性较GmKNOXl类低,表达部位更广泛。     

大豆KNOX基因家族的结构和表达分析

KNOX基因家族编码同源异型盒蛋白, 在植物生长发育过程中起重要调控作用。利用生物信息学手段在全基因组水平上对大豆(Glycine max)KNOX家族基因进行鉴定和分类, 并分析其基因结构、蛋白同源结构域特征以及基因表达方式。研究结果表明: 大豆中的27个GmKNOX基因可以分为GmKNOX I和GmKNOX II两个亚类, 其中GmKNOX I类可分为3个主要的进化支, GmKNOX II类分为2个主要的进化支; 26个GmKNOX基因不均匀地分布在16条染色体上, GmKNOX27尚无法定位。不同组织表达谱的分析表明: GmKNOX I类基因表达部位比较集中, 以茎顶端分生组织中表达量最高; 而GmKNOX II类基因的表达特异性较GmKNOX I类低, 表达部位更广泛。     

Overexpression of Arabidopsis ACK1 alters leaf morphology and retards growth and development

Cyclin dependent kinases (CDKs) play important roles in the plant cell cycle, a highly coordinated process in plant growth and development. To understand the regulatory network involving the CDKs, we have examined the role of ACK1, a gene that has significant homology to known ICKs (inhibitors of CDKs), but occupies a distinct branch of the ICK phylogenetic tree. Overexpression of ACK1 in transgenic Arabidopsis significantly inhibited growth, leading to effects such as serration of leaves, as a result of strong inhibition of cell division in the leaf meristem. ACK1 transgenic plants also differed morphologically from control Arabidopsis plants, and the cells of ACK1 transgenics were more irregular than the corresponding cells of control plants. These results suggest that ACK1 acts as a CDK inhibitor in Arabidopsis, and that the alterations in leaf shape may be the result of restricted cell division.     

Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis

The WNK (With No Lysine K) serine-threonine kinases have been shown to be osmosensitive regulators and are critical for cell volume homeostasis in humans. We previously identified a soybean root-specific WNK homolog, GmWNK1, which is important for normal late root development by fine-tuning regulation of ABA levels. However, the functions of WNKs in plant osmotic stress response remains uncertain. In this study, we generated transgenic Arabidopsis plants with constitutive expression of GmWNK1. We found that these transgenic plants had increased endogenous ABA levels and altered expression of ABA-responsive genes, and exhibited a significantly enhanced tolerance to NaCl and osmotic stresses during seed germination and seedling development. These findings suggest that, in addition to regulating root development, GmWNK1 also regulates ABA-responsive gene expression and/or interacts with other stress related signals, thereby modulating osmotic stress responses. Thus, these results suggest that WNKs are members of an evolutionarily conserved kinase family that modulates cellular response to osmotic stresses in both animal and plants.     

种子特异表达异源DGAT1基因提高大豆种子含油量和营养品质

为提高大豆Glycine max种子含油量和营养品质,文中以二酰甘油酰基转移酶1(Diacylglycerol acyltransferase 1,DGAT1)基因为遗传修饰靶标。将来自高油植物斑鸠菊Vernonia galamensis L.编码DGAT1酶蛋白的c DNA克隆Vg DGAT1A在大豆种子特异超表达。连续选择获得高代(T7)Vg DGAT1A转基因大豆株系。转基因株系表型鉴定显示,在大豆种子发育中期(30–45 DAF),Vg DGAT1A高表达,相应地DGAT酶活性是非转基因野生型和空载体转化对照的7.8倍。转基因成熟种子含油量比对照提高了5.1%,淀粉含量比对照减少2%–3%,蛋白质含量与对照无显著差异。此外,转基因大豆种子百粒重(14.5 g)和种子萌发率(95.6%)与对照亦无明显差异。种子油脂脂肪酸成分分析显示,转基因大豆种子油中抗氧化的油酸(C18:1Δ9)含量比对照提高8.2%,相应地易氧化的亚油酸(C18:2Δ9,12)和亚麻酸(C18:3Δ9,12,15)分别减少6%和2%。这些数据表明,种子特异超表达外源Vg DGAT1A基因,打破了大豆种子含油量和蛋白质含量的负连锁,显著提高种子含油量且未导致蛋白含量降低。转基因大豆种子重量和萌发率亦未显负效应,而且种子油脂抗氧化性和营养品质得以改善。研究表明应用这一高酶活性Vg DGAT1A的基因工程是提高种子含油量和改善油脂品质的一条有效途径。     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

Expression of new mutant alleles of AS1 and AS2 genes controlling leaf morphogenesis in Arabidopsis thaliana

We have studied the morphology and vein branching of rosette leaves in Arabidopsis thaliana mutants as and sa, which proved to be alleles of the A.thaliana AS1 and AS2 genes, respectively. We have also analyzed the localization of bioactive auxin, as measured by the expression of the DR5::GUS transgene, as well as the expression patterns of BP, as measured by the expression of the BP::GUS transgene in leaves of the mutants. In mature leaves of the mutants, BP was expressed ectopically. Furthermore, the mutants showed some defects in the localization and concentration of free auxin compared to the wild type. Our results of studying new alleles of AS1 and AS2 support their role in control of class I KNOX genes and auxin transport.