Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield

Vigna Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na(+) was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.

甘蓝型油菜中脯氨酸积累的分子调控机制

　　渗透胁迫下植物中游离脯氨酸的积累被普遍认为对植物有保护作用。本文对甘蓝型油菜（Brassica napus）中的脯氨酸积累及其调控机理进行了研究。正常生长条件下幼苗叶中脯氨酸含量最高，根中脯氨酸含量最低，盐胁迫后不同部位脯氨酸积累程度相近。盐浓度200 mM起，幼苗开始有明显脯氨酸积累,并随着浓度的增加而增加。PEG处理后脯氨酸积累要明显高于ABA和盐胁迫处理后脯氨酸积累。在成株中，生殖器官中脯氨酸含量明显高于营养器官中脯氨酸含量。 　　我们克隆了编码Δ1-二氢吡咯啉-5-羧酸合成酶（P5CS）,鸟氨酸转氨酶(OAT)和脯氨酸脱氢酶(PDH)的四个基因的cDNA。通过Southern杂交检测P5CS基因在甘蓝型油菜及其亲本白菜型油菜和甘蓝中的拷贝数，发现杂交条带在4倍体油菜中并没有显著的多于其两个2倍体亲本，推测有可能是在物种进化的过程中发生了基因丢失。 　　利用实时定量PCR的方法检测了渗透胁迫下甘蓝型油菜中BnP5CS，BnOAT和BnPDH等基因的表达水平。在ABA处理，盐处理和干旱处理时，BnP5CS1和BnP5CS2的表达在脯氨酸积累开始前就开始有明显上调，但是BnP5CS1的上调水平要比BnP5CS2高许多。BnOAT在ABA处理后表达水平没有太大的变化，在盐胁迫后甚至有一点下调，在干旱处理后却表现为一定程度的上调。另一方面，BnPDH的表达在ABA处理，盐胁迫和干旱胁迫后都受到了抑制。在成株不同器官相关基因表达的研究中发现，BnOAT在叶中表达量最高，BnP5CS和BnPDH在花蕾和花中的表达水平都比其他器官中要高。 　　我们的结果说明，渗透胁迫下甘蓝型油菜中的脯氨酸积累是脯氨酸合成途径的诱导和脯氨酸降解途径的抑制共同作用的结果。在轻度的渗透胁迫下，谷氨酸合成途径占主导地位，而在较为严重的渗透胁迫后期，谷氨酸合成途径和鸟氨酸合成途径共同起作用。甘蓝型油菜生殖器官花和花蕾中脯氨酸的代谢非常旺盛，说明脯氨酸可能在花发育中起着一定的作用。

Expressão gênica diferencial em palmitos de cana-de-açúcar submetida a diferentes períodos de estresse hídrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Hypersensitivity of an Arabidopsis Sugar Signaling Mutant toward Exogenous Proline Application

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Δ1-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutantrsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS andP5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Δ1-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.

Comparative Analysis of the Regulation of Expression and Structures of Two Evolutionarily Divergent Genes for Δ1-Pyrroline-5-Carboxylate Synthetase from Tomato1

We isolated two tomato (Lycopersicon esculentum) cDNA clones, tomPRO1 and tomPRO2, specifying Δ1-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of proline (Pro) biosynthesis. tomPRO1 is unusual because it resembles prokaryotic polycistronic operons (M.G. García-Ríos, T. Fujita, P.C. LaRosa, R.D. Locy, J.M. Clithero, R.A. Bressan, L.N. Csonka [1997] Proc Natl Acad Sci USA 94: 8249–8254), whereas tomPRO2 encodes a full-length P5CS. We analyzed the accumulation of Pro and the tomPRO1 and tomPRO2 messages in response to NaCl stress and developmental signals. Treatment with 200 mm NaCl resulted in a >60-fold increase in Pro levels in roots and leaves. However, there was a <3-fold increase in the accumulation of the tomPRO2 message and no detectable induction in the level of the tomPRO1 message in response to NaCl stress. Although pollen contained approximately 100-fold higher levels of Pro than other plant tissues, there was no detectable increase in the level of either message in pollen. We conclude that transcriptional regulation of these genes for P5CS is probably not important for the osmotic or pollen-specific regulation of Pro synthesis in tomato. Using restriction fragment-length polymorphism mapping, we determined the locations of tomPRO1 and tomPRO2 loci in the tomato nuclear genome. Sequence comparison suggested that tomPRO1 is similar to prokaryotic P5CS loci, whereas tomPRO2 is closely related to other eukaryotic P5CS genes.