Pei Hou

Expression of an Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+-PPase from Arabidopsis may result in a higher proton electrochemical gradient, which facilitates enhanced sequestering of ions and sugars into the vacuole, reducing water potential and resulting in increased drought- and salt tolerance when compared to wild-type plants. Furthermore, overexpression of AVP1 stimulates auxin transport in the root system and leads to larger root systems, which helps transgenic plants absorb water more efficiently under drought conditions. Using the same approach, AVP1-expressing cotton plants were created and tested for their performance under high-salt and reduced irrigation conditions. The AVP1-expressing cotton plants showed more vigorous growth than wild-type plants in the presence of 200 mM NaCl under hydroponic growth conditions. The soil-grown AVP1-expressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in greenhouse conditions. Furthermore, the fibre yield of AVP1-expressing cotton plants is at least 20% higher than that of wild-type plants under dry-land conditions in the field. This research indicates that AVP1 has the potential to be used for improving crops drought- and salt tolerance in areas where water and salinity are limiting factors for agricultural productivity.

A ribosome-inactivating protein (curcin 2) induced from Jatropha curcas can reduce viral and fungal infection in transgenic tobacco

A new kind of ribosome-inactivating protein (curcin 2), induced by several different kinds of stress from Jatropha curcas leaves, under the control of the CaMV (cauliflower mosaic virus) 35S promoter, was introduced into the tobacco genome by Agrobacterium tumefaciens-mediated transformation method. The curcin 2 protein was only detected in the transgenic tobacco plantlets transformed with the cur2p fragment (coding premature curcin 2 protein), but not in the plantlets with the cur2m fragment (coding mature curcin 2 protein). The T1 population of the transgenic lines shows an increased tolerance to tobacco mosaic virus (TMV) and a fungal pathogen Rhizoctonia solani by delaying the development of systemic symptoms of TMV and reducing the damage caused by the fungal disease. The increases of the tolerances correspond to the curcin 2 level in the transgenic plants.

Both PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2 Promote Seedling Photomorphogenesis in Multiple Light Signaling Pathways

Both PAR1 and PAR2, encoding bHLH transcription factors, enhance seedling deetiolation under far-red, red, and blue light conditions. Arabidopsis (Arabidopsis thaliana) seedlings undergo photomorphogenesis in the light and etiolation in the dark. Light-activated photoreceptors transduce the light signals through a series of photomorphogenesis promoting or repressing factors to modulate many developmental processes in plants, such as photomorphogenesis and shade avoidance. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a conserved RING finger E3 ubiquitin ligase, which mediates degradation of several photomorphogenesis promoting factors, including ELONGATED HYPOCOTYL5 (HY5) and LONG HYPOCOTYL IN FAR-RED1 (HFR1), through a 26S proteasome-dependent pathway. PHYTOCHROME RAPIDLY REGULATED1 (PAR1) was first detected as an early repressed gene in both phytochrome A (phyA)-mediated far-red and phyB-mediated red signaling pathways, and subsequent studies showed that both PAR1 and PAR2 are negative factors of shade avoidance in Arabidopsis. However, the role of PAR1 and PAR2 in seedling deetiolation, and their relationships with other photomorphogenesis promoting and repressing factors are largely unknown. Here, we confirmed that both PAR1 and PAR2 redundantly enhance seedling deetiolation in multiple photoreceptor signaling pathways. Their transcript abundances are repressed by phyA, phyB, and cryptochrome1 under far-red, red, and blue light conditions, respectively. Both PAR1 and PAR2 act downstream of COP1, and COP1 mediates the degradation of PAR1 and PAR2 through the 26S proteasome pathway. Both PAR1 and PAR2 act in a separate pathway from HY5 and HFR1 under different light conditions, except for sharing in the same pathway with HFR1 under far-red light. Together, our results substantiate that PAR1 and PAR2 are positive factors functioning in multiple photoreceptor signaling pathways during seedling deetiolation.

Different functions and expression profiles of curcin and curcin-L in Jatropha curcas L.

To date, two types of ribosome-inactivating proteins (RIPs) have been found in Jatropha curcas. One is curcin, which has been isolated from the endosperm, and the other is curcin-L, which is expressed in leaves upon stress treatment. Phylogenetic analysis of the predicted amino acid sequences of the RIPs in plants revealed that these belong to a major subfamily and are close to trichosanthin (TCS). Studies on the mRNA and protein levels showed that both curcin and curcin-L have an organ-specific expression pattern. Curcin is only expressed and accumulated in the endosperm; its expression begins in the globular embryo period and peaks during the mature embryo period. In contrast, curcin-L is only expressed in the leaves, but its expression is induced by certain conditions such as treatment with phytohormones or polyethylene glycol, exposure to high and low temperatures, and fungal infection. Analysis of the 5’ flanking regions of curcin and curcin-L revealed that the 5’ flanking region of curcin-L has three major inserted fragments, which are not present in the corresponding region of curcin. Comparison of characteristic cis-elements suggests the presence of several motifs that are involved in the endosperm-specific expression in the 5’ flanking region of curcin, while in curcin-L some stress- and defense-responsive motifs are found to be mainly located in the three inserted fragments. Comparison of the antifungal activity of the two RIPs showed that the one of curcin-L is higher than that of curcin. Differences in the expression and activity of curcin and curcin-L suggest that these two RIPs have different functions.

Overexpressing a novel RING-H2 finger protein gene, OsRHP1, enhances drought and salt tolerance in rice (Oryza sativa L.)

RING (really interesting new gene) zinc-finger proteins have important regulatory roles in the development of a variety of organisms. In the present report, a full length cDNA encoding a novel RING-H2 finger protein from rice, designated as OsRHP1, was isolated and characterized. OsRHP1 encodes a small protein (167 amino acids) with two N-terminal trans-membrane domains and a canonical RING-H2 zinc-finger motif located at the C-terminus. Ten putative homologs of OsRHP1 harboring one canonical RING-H2 finger domain exhibit in other plant species. In our work, OsRHP1 was expressed ubiquitously in various tissues, but its transcript is accumulated more in mature leaf. Compared with wild-type (WT) rice plants, transgenic plants overexpressing OsRHP1 exhibited more tolerance to drought and salt stress. Meanwhile, the transgenic T2 lines showed a significant increase of endogenous ABA content in normal condition. When subjected to drought and salt stress, much stronger accumulation of ABA was detected compared with WT. Furthermore, a significant increase of transcriptional expression, of five ABA biosynthesis or ABA-mediated response genes, OsNCED, OsZEP, OsAAO, OsABI5, OsABF2, OsLEA3-1 were observed in the transgenic plants under drought and salt stress condition. These results demonstrate that overexpression of OsRHP1 substantially enhances drought and salt tolerance through increased ABA level and enhanced ABA-mediated stress response.

Overexpression of Arabidopsis XERICO gene confers enhanced drought and salt stress tolerance in rice (Oryza Sativa L.)

Drought and salinity are two major limiting factors in rice (Oryza. sativa L.) productivity worldwide. XERICO, encoding a RING-H2 zinc finger protein, substantially enhanced drought tolerance by increasing ABA biosynthesis in Arabidopsis. Here, we report the isolation of the full-length cDNA of XERICO from Arabidopsis and its heterologous expression in rice (O. sativa L.). Homozygous transgenic plants overexpressing XERICO (OE-XERICO) exhibited hypersensitivity to stress stimuli (salt, osmotic stress and exogenous ABA) during seed germination and early seedling growth. When subjected to dehydration and salinity stress, 4-week-old transgenic seedlings showed a considerable increase in tolerance to these stimuli compared with that of the wild type (WT) seedlings. Detached leaves from the transgenic rice lines showed lower transpirational water loss than WT. The OE-XERICO lines exhibited a significant increase in endogenous ABA contents, and expression levels of Four ABA biosynthesis or ABA-response genes, OsNCED, OsABA3, OsABI5, OsLEA3-1, under drought and salt stress condition. These results suggest that the overexpression of XERICO in rice plants confers improved drought and salt tolerance probably through enhanced ABA level and ABA-mediated stress response.

Authentication of Angelica anomala Avé-Lall cultivars through DNA barcodes

Angelica anomala Avé-Lall (Chuanbaizhi in Chinese) is an important medicinal plant which can be used in traditional Chinese medicines; however, there are no authentic and universal methods to differentiate this Sichuan famous-region drug of A. anomala from a large number of non-famous-region and false drugs. It has been demonstrated that DNA barcoding is a molecular diagnostic method for species identification, which uses a single standardized DNA fragment. In this study, we tested five DNA barcoding candidates (matK, ITS, ITS2, rbcL, and psbA-trnH), and we found that ITS was the best candidate to authenticate the famous-region drug of A. anomala. Moreover, through comparative analysis of these five DNA barcodes between A. anomala and Angelica dahurica, we found that ITS had the most and ITS2 had more variable regions, but the psbA-trnH, rbcL, and matK regions were identical. Hence, we suggest ITS as the DNA barcoding to identify A. anomala and A. dahurica. Moreover, we are determined to adopt the A. anomala as the accurate Latin name of Chuanbaizhi.

Knockout of the VPS22 component of the ESCRT-II complex in rice ( Oryza sativa L.) causes chalky endosperm and early seedling lethality

In both yeast and mammals, the major constituent of the endosomal sorting complex required for transport-II (ESCRT-II) is the VPS22/EAP30 protein, which plays an important role in ubiquitin-mediated degradation of membrane proteins through the multivesicular body pathway. However, the functions of ESCRT-II subunits in plants are largely unknown. In this work, we report the genetic analysis and phenotypic characterization of mutants in OsVPS22 gene, which encodes a functional VPS22 homolog in rice. On the basis of a collection of T-DNA lines, we identified a T-DNA insertion mutant, which showed abnormal segregation ratios; we then found that the T-DNA insertion is located within the sixth intron of the OsVPS22 gene. Compared with the wild type, this vps22 mutant exhibited seedling lethality and severe reduction in shoot and root growth. In addition, the vps22 mutant had a chalky endosperm in the grain. In summary, our data suggest that OsVPS22 may be required for seedling viability and grain filling in rice, thus providing a valuable resource for further exploration of the functions of the ESCRTing machinery in plants.

Synergistic and Antagonistic Action of Phytochrome (Phy) A and PhyB during Seedling De-Etiolation in Arabidopsis thaliana

It has been reported that Arabidopsis phytochrome (phy) A and phyB are crucial photoreceptors that display synergistic and antagonistic action during seedling de-etiolation in multiple light signaling pathways. However, the functional relationship between phyA and phyB is not fully understood under different kinds of light and in response to different intensities of such light. In this work, we compared hypocotyl elongation of the phyA-211 phyB-9 double mutant with the wild type, the phyA-211 and phyB-9 single mutants under different intensities of far-red (FR), red (R), blue (B) and white (W) light. We confirmed that phyA and phyB synergistically promote seedling de-etiolation in B-, B plus R-, W- and high R-light conditions. The correlation of endogenous ELONGATED HYPOCOTYL 5 (HY5) protein levels with the trend of hypocotyl elongation of all lines indicate that both phyA and phyB promote seedling photomorphogenesis in a synergistic manner in high-irradiance white light. Gene expression analyses of RBCS members and HY5 suggest that phyB and phyA act antagonistically on seedling development under FR light.

Molecular cloning, characterization, and expression of an omega-3 fatty acid desaturase gene from Sapium sebiferum

A full-length cDNA (SsFAD3) for an omega-3 fatty acid desaturase (omega-3 FAD) was cloned from Sapium sebiferum (L.) Roxb. using rapid amplification of cDNA ends and reverse transcription polymerase chain reaction methods. SsFAD3 contained a 1119-bp open reading frame encoding a 372-amino acid polypeptide. The genomic sequence region of the SsFAD3 ORF was composed of 8 exons and 7 introns, similar to other omega-3 FADs found in most plants. The amino acid sequence showed a higher identity with microsomal omega-3 FADs than plastidial omega-3 FADs. Southern blot analysis of SsFAD3 suggested the existence of a small gene family composed of several copies or closely linked genes. SsFAD3 transcripts were detected in shoots, roots, leaves, stems, and seeds, but were most abundant in shoots. The function of SsFAD3 was confirmed by the accumulation of alpha-linolenic acid (alpha-18:3) in Saccharomyces cerevisiae transformants.