Physiological and transcriptome analyses reveal tissue-specific responses of Leucaena plants to drought stress.

Leucaena leucocephala (Leucaena) is a leguminous tree widely cultivated in tropical and subtropical regions due to its strong environmental suitability for abiotic stresses, especially drought. However, the molecular mechanisms and key pathways involved in Leucaena's drought response require further elucidation. Here, we comparatively analyzed the physiological and early transcriptional responses of Leucaena leaves and roots under drought stress simulated by polyethylene glycol (PEG) treatments. Drought stress induced physiological changes in Leucaena seedlings, including decreases in relative water content (RWC) and increases in relative electrolyte leakage (REL), malondialdehyde (MDA), proline contents as well as antioxidant enzyme activities. In response to drought stress, 6461 and 8295 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. In both tissues, the signaling transduction pathway of plant hormones was notably the most enriched. Specifically, abscisic acid (ABA) biosynthesis and signaling related genes (NCED, PP2C, SnRK2 and ABF) were strongly upregulated particularly in leaves. The circadian rhythm, DNA replication, alpha-linolenic acid metabolism, and secondary metabolites biosynthesis related pathways were repressed in leaves, while the glycolysis/gluconeogenesis and alpha-linolenic acid metabolism and amino acid biosynthesis processes were promoted in roots. Furthermore, heterologous overexpression of Leucaena drought-inducible genes (PYL5, PP2CA, bHLH130, HSP70 and AUX22D) individually in yeast increased the tolerance to drought and heat stresses. Overall, these results deepen our understanding of the tissue-specific mechanisms of Leucaena in response to drought and provide target genes for future drought-tolerance breeding engineering in crops.

Characterization of a gene encoding clathrin heavy chain in maize up-regulated by salicylic acid, abscisic acid and high boron supply.

Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA, suggesting that ZmCHC1 gene may be involved in the SA signaling pathway in maize defense responses. The expressions of ZmCHC1 and ZmCHC2 genes in maize are down-regulated by azide or cold treatment, further revealing the energy requirement of CME and suggesting that CME in plants is sensitive to low temperatures.

[Promoter specificity of LE-ACS6 gene in LE-ACS6::GUS transgenic arabidopsis plants].

The LE-ACS6 gene encodes ACC synthase, the key enzyme of ethylene biosynthesis pathway. Accumulation of LE-ACS6 transcripts is concomitant with the system 1 ethylene production in the pre-climacteric tomato fruit, and both are down regulated by exogenous ethylene treatment. To elucidate the possible role of system 1 ethylene in plant development and investigate the promoter tissue specificity of LE-ACS6 gene, stable transformation of Arabidopsis with a LE-ACS6 promoter-GUS fusion construct by Agrobacterium method has been done. Histochemical localization of GUS activity and beta-glucuronidase enzyme assay in transgenic LE-ACS6::GUS plants showed strong expression of GUS in cotyledons and hypocotyls of 6 d seedlings, but no GUS activity was detected in roots. The GUS activity of 6 d seedling was increased significantly when treated with NAA 10(-4)mol/L. In 40 d rosette leaf LE-ACS6 promoter driven GUS gene was predominantly expressed in mature leaves. Lower level of GUS expression was detected in younger and older leaves. Wounding was found to increase GUS gene expression in transgenic leaves. Again, exogenous NAA was found to increase the GUS activity in wounded leaf tissue. Strong staining reaction was observed in the top part of rapidly growing stems. In different developmental stages of Arabidopsis seeds, "mature green" pods were strongly stained, but "ripening fruits" were not colored. These observations are concomitant with the system 1 ethylene production, suggesting a popular mechanism in regulating ethylene biosynthesis in different plants, at least in tomato and Arabidopsis.

Cloning and expression analysis of cDNAs encoding ABA 8'-hydroxylase in peanut plants in response to osmotic stress.

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic-stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by ABA 8'-hydroxylase, the cytochrome P450 CYP707A family. In this study, the full-length cDNAs of AhCYP707A1 and AhCYP707A2 were cloned and characterized from peanut. Expression analyses showed that AhCYP707A1 and AhCYP707A2 were expressed ubiquitously in peanut roots, stems, and leaves with different transcript accumulation levels, including the higher expression of AhCYP707A1 in roots. The expression of AhCYP707A2 was significantly up-regulated by 20% PEG6000 or 250 mmol/L NaCl in peanut roots, stems, and leaves, whereas the up-regulation of AhCYP707A1 transcript level by PEG6000 or NaCl was observed only in roots instead of leaves and stems. Due to the osmotic and ionic stresses of high concentration of NaCl to plants simultaneously, low concentration of LiCl (30 mmol/L, at which concentration osmotic status of cells is not seriously affected, the toxicity of Li+ being higher than that of Na+) was used to examine whether the effect of NaCl might be related to osmotic or ionic stress. The results revealed visually the susceptibility to osmotic stress and the resistance to salt ions in peanut seedlings. The significant up-regulation of AhCYP707A1, AhCYP707A2 and AhNCED1 transcripts and endogenous ABA levels by PEG6000 or NaCl instead of LiCl, showed that the osmotic stress instead of ionic stress affected the expression of those genes and the biosynthesis of ABA in peanut. The functional expression of AhCYP707A1 cDNA in yeast showed that the microsomal fractions prepared from yeast cell expressing recombinant AhCYP707A1 protein exhibited the catalytic activity of ABA 8'-hydroxylase. These results demonstrate that the expressions of AhCYP707A1 and AhCYP707A2 play an important role in ABA catabolism in peanut, particularly in response to osmotic stress.

Regulation of ABA level and water-stress tolerance of Arabidopsis by ectopic expression of a peanut 9-cis-epoxycarotenoid dioxygenase gene.

The oxidative cleavage of cis-epoxycarotenoids catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is considered to be the rate-limiting step in abscisic acid (ABA) biosynthesis. Here we demonstrate that the expression of AhNCED1 gene in peanut plants is significantly up-regulated by dehydration and high salinity. The AhNCED1 transcript and endogenous ABA both accumulate predominantly in leaves and stems of peanut in response to dehydration. The considerable up-regulation of AhNCED1 expression by exogenous application of ABA suggests a positive feedback control of ABA biosynthesis in peanut. NAA at high concentration increases ABA biosynthesis in peanut plants through up-regulation of the AhNCED1 gene expression. The constitutive expression of the AhNCED1 gene in wild-type Arabidopsis results in an increase of ABA accumulation in transgenic plants in response to drought stress. Ectopic expression of AhNCED1 gene in 129B08/nced3 mutant Arabidopsis (with impaired AtNCED3 gene involved in ABA biosynthesis under water stress) driven by the AtNCED3 promoter restores its ability to accumulate ABA during drought stress, and reverts its hypersensitivity to nonionic osmotic stress and soil drought. These results indicate that the expression of AhNCED1 gene plays an important role in the regulation of ABA level during water stress, and that water-stress tolerance of Arabidopsis plants can be improved by ectopic expression of the AhNCED1 gene causing accumulation of endogenous ABA.