Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence

Many plant genes have been shown to be induced by water stress and function in stress tolerance. The erd1 gene has been shown to be upregulated in response to both water stress and etiolation. Promoter studies using the erd1 promoter region fused to the luciferase (LUC) reporter gene in Arabidopsis thaliana were performed to identify the putative cis elements involved. Results indicated that the cis elements, responsible for gene expression during dehydration and etiolation, are separately located in two discrete portions of the erd1 promoter. Base substitution analysis showed that a 14-bp region from -599 to -586, and a myc recognition motif from -466 to -461 are necessary for the induction of LUC activity in dehydrated plants. On the other hand, base substitution analysis revealed that both an abscisic acid responsive element (ABRE)-like sequence (from -199 to -195) and an ACGT sequence (from -155 to -152) are required for an etiolation-induced increase in LUC activity. LUC activity measurements from etiolated transgenic plants incubated in either water, N6-benzyleadenine (BA), or a 1% sucrose solution found that while BA was able to delay the increase in LUC activity seen in water-treated plants, no increase in LUC activity was seen in plants incubated in sucrose. These results indicate that the erd1 promoter contains two different regulatory systems that are involved in upregulation by dehydration stress and dark-induced senescence.     

Cloning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) inArabidopsis thaliana L.: identification of three ERDs as HSP cognate genes

InArabidopsis thaliana L., accumulation of abscisic acid (ABA) began to increase 2 h after plants had been subjected to dehydration stress and reached maximum levels after 10h. Differential hybridization was used to isolate 26Arabidopsis cDNAs with gene expression induced by a 1 h dehydration treatment. The cDNA clones were classified into 16 groups based on Southern blot hybridization, and named ERD (early-responsive todehydration) clones. Partial sequencing of the cDNA clones revealed that three ERDs were identical to those of HSP cognates (Athsp70-1, Athsp81-2, and ubiquitin extension protein). Dehydration stress strongly induced the expression of genes for the three ERDs, while application of ABA, which is known to act as a signal transmitter in dehydration-stressed plants, did not significantly affect the ERD gene expression. This result suggests that these HSP cognates are preferentially responsive to dehydration stress inA. thaliana, and that signaling pathways for the expression of these genes under conditions of dehydration stress are not mainly mediated by ABA. We also discuss the possible functions of these three ERD gene products against dehydration stress.     

Dithiothreitol increases b-glucuronidase accumulation in transformed tobacco (Nicotiana tabacum) protoplasts without altering their viability or the synthesis and export of cellular proteins

The effect of dithiothreitol (DTT) on the expression of the β-glucuronidase (GUS) reporter gene under the control of the CaMV-35 S promoter has been investigated by radioactive labelling and immunoprecipitation of the enzyme in protoplasts from stably transformed tobacco plants and compared with that observed in protoplasts transiently expressing the same gene construct. An increase in net accumulation of GUS during the culture period in response to externally added DTT (2 mm) was observed both in protoplasts from transformed tobacco plants and in electroporated protoplasts. DTT had no effect on rate of degradation of the mature GUS protein, as shown in a pulse-chase experiment. Relevant aspects of protoplast physiology, such as viability, synthesis of ³⁵S-labelled cellular proteins, or synthesis and export of pathogenesis-related proteins (one putative chitinase and two 1,3-β-glucanases) were not affected by the reducing reagent. Received: 15 December 1997 / Revision received: 14 April 1998 / Accepted: 1 May 1998     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

拟南芥ERD15基因缺失突变体的分子鉴定和表型分析

为探究ERD15基因功能,利用反向遗传学,通过PCR及半定量PCR筛选鉴定出拟南芥(Arabidopsis thaliana) ERD15基因的T-DNA插入纯合突变体,并对其表型进行观察分析。结果表明,erd15突变体莲座叶数目显著增多,提前3～4 d开花,突变体比野生型更早从营养生长转向生殖生长。拟南芥野生型植株主茎为圆柱体,平均直径1.29 mm,而erd15突变体主茎扁平,平均直径达到2.27mm,具极显著差异。与野生型相比,erd15突变体果实心皮发育受到影响,隔膜上排列有多排种子,果荚顶端膨大,长度缩短37.67%,但角果平均结籽数升高。因此,ERD15基因参与了调控拟南芥植株的生殖生长过程。     

Identification of clp genes expressed in senescing Arabidopsis leaves.

Clp protease is a highly selective protease in E. coli, which consists of two types of subunits, the regulatory subunit with ATPase activity, ClpA, and the catalytic subunit, ClpP. In order to examine the possible association of plant Clp protease with the degradation of protein in senescing chloroplasts, we isolated a cDNA clone for ClpC which is a plant homologue of ClpA from Arabidopsis thaliana in addition to ERD1 which we had isolated earlier [Kiyosue et al. (1993) Biochem. Biophys. Res. Commun. 196: 1214]. We also isolated a clone for the plastidic gene, clpP (pclpP) and cDNA clones for putative nuclear clpP genes (nclpP1-6). We analyzed the expression of these clp genes in Arabidopsis leaves after various dark periods and during natural senescence. The expression of erd1 was increased by dark-induced and by natural senescence, as reported earlier [Nakashima et al. (1997) Plant J. 12: 851], while that of AtclpC was decreased. Two catalytic subunits nclpPs (nclpP3 and nclpP5) showed high expression in naturally senescing leaves, but the expression of pclpP and the other nclpPs was not changed. Immunoblot analysis of chloroplast protein and in vitro import analysis demonstrated that both nucleus-encoded regulatory subunits as well as nClpP5 were localized in the chloroplast stroma. These observations suggest that chloroplast Clp protease is composed of very complicated combinations of subunits, and that ERD1, nClpP5 and pClpP have a role in the concerted degradation of protein in senescing chloroplasts.     

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T₂ generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.     

Evidence for contribution of autophagy to Rubisco degradation during leaf senescence in Arabidopsis thaliana

During leaf senescence, Rubisco is gradually degraded and its components are recycled within the plant. Although Rubisco can be mobilized to the vacuole by autophagy via specific autophagic bodies, the importance of this process in Rubisco degradation has not been shown directly. Here, we monitored Rubisco autophagy during leaf senescence by fusing synthetic green fluorescent protein (sGFP) or monomeric red fluorescent protein (mRFP) with Rubisco in Arabidopsis (Arabidopsis thaliana). When attached leaves were individually exposed to darkness to promote their senescence, the fluorescence of Rubisco‐sGFP was observed in the vacuolar lumen as well as chloroplasts. In addition, release of free‐sGFP due to the processing of Rubisco‐sGFP was observed in the vacuole of individually darkened leaves. This vacuolar transfer and processing of Rubisco‐sGFP was not observed in autophagy‐deficient atg5 mutants. Unlike sGFP, mRFP was resistant to proteolysis in the leaf vacuole of light‐grown plants. The vacuolar transfer and processing of Rubisco‐mRFP was observed at an early stage of natural leaf senescence and was also obvious in leaves naturally covered by other leaves. These results indicate that autophagy contributes substantially to Rubisco degradation during natural leaf senescence as well as dark‐promoted senescence.     

Gene regulation in intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum tissue protoplasts

Activities of the ß-glucuronidase (GUS) reporter enzyme were evaluated in transgenic plants, protoplasts, and intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum. With GUS under control of the promoter of the cauliflower-mosaicvirus 35S RNA gene (CaMV), activities of the enzyme were nearly evenly distributed over the tissues of plants grown in vitro. Activities in microtubers of potato plants with the reporterenzyme under control of the promoter of granule-bound starch synthase (GBSS) from S. tuberosum were higher than in leaves. The CaMV-GUS construct present in leaf protoplasts showed an increased expression in biochemical and cytochemical assays after fusion with wildtype-tuber protoplasts. Such an increase was not observed in the case of the GBSS-GUS constructs. It was concluded that nuclei in plant heterokaryons are being influenced by the fusion partner, but that repressive, trans-acting regulation factors in leaf protoplasts possibly prevent an increase of the expression of the chimeric GBSS-gene in heterokaryons.     

Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic beta-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.     

拟南芥莲座叶片发育过程中P1基因的表达特性

该实验对CDF1类似蛋白基因(P1)在拟南芥叶片发育不同阶段的定量PCR结果显示,P1基因在拟南芥叶片发育的所有时期均可表达,但在茎生叶和衰老叶中的表达水平明显高于成熟叶和幼叶。GUS报告基因表达的组织化学染色结果显示,P1启动子在拟南芥叶片中有较高的驱动活性;在营养生长阶段的幼苗和植株(4~5周)的所有叶片中均能检测到GUS表达,但在植株转入生殖生长阶段后(6周及以后),GUS表达主要集中在逐渐衰老的叶中,并随着叶片衰老程度加剧GUS染色程度也越深,这一结果与GUS荧光定量检测结果一致。通过分析P1基因启动子上可能存在的顺式调控元件,发现茉莉酸甲酯、热压、干旱和水杨酸等均能够引起叶片衰老调控元件的响应,证实P1的表达受到这些因素的调控。研究表明,P1在拟南芥莲座叶片中很可能参与了对上游衰老信号的响应,该研究结果为进一步探究P1在叶片衰老过程中的分子功能验证奠定了基础。     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.