Investigation of the AQP Family in Soybean and the Promoter Activity of TIP2;6 in Heat Stress and Hormone Responses.

Aquaporins (AQPs) are one diverse family of membrane channel proteins that play crucial regulatory roles in plant stress physiology. However, the heat stress responsiveness of AQP genes in soybean remains poorly understood. In this study, 75 non-redundant AQP encoding genes were identified in soybean. Multiple sequence alignments showed that all GmAQP proteins possessed the conserved regions, which contained 6 trans-membrane domains (TM1 to TM6). Different GmAQP members consisted of distinct Asn-Pro-Ala (NPA) motifs, aromatic/arginine (ar/R) selectivity filters and Froger's positions (FPs). Phylogenetic analyses distinguished five sub-families within these GmAQPs: 24 GmPIPs, 24 GmTIPs, 17 GmNIPs, 8 GmSIPs, and 2 GmXIPs. Promoter cis-acting elements analyses revealed that distinct number and composition of heat stress and hormone responsive elements existed in different promoter regions of GmAQPs. QRT-PCR assays demonstrated that 12 candidate GmAQPs with relatively extensive expression in various tissues or high expression levels in root or leaf exhibited different expression changes under heat stress and hormone cues (abscisic acid (ABA), l-aminocyclopropane-l-carboxylic acid (ACC), salicylic acid (SA) and methyl jasmonate (MeJA)). Furthermore, the promoter activity of one previously functionally unknown AQP gene-GmTIP2;6 was investigated in transgenic Arabidopsis plants. The beta-glucuronidase (GUS) activity driven by the promoter of GmTIP2;6 was strongly induced in the heat- and ACC-treated transgenic plants and tended to be accumulated in the hypocotyls, vascular bundles, and leaf trichomes. These results will contribute to uncovering the potential functions and molecular mechanisms of soybean GmAQPs in mediating heat stress and hormone signal responses.

Development and characterization of 41 novel EST-SSR markers for Pisum sativum (Leguminosae).

PREMISE OF THE STUDY: Expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers were developed in Pisum sativum for further use in genetic studies and breeding programs. METHODS AND RESULTS: Forty-one novel EST-SSR primers were developed and characterized for size polymorphism in 32 Pisum sativum individuals from four populations from China. In each population, the number of alleles per locus ranged from one to seven, with observed heterozygosity and expected heterozygosity ranging from 0 to 0.8889 and 0 to 0.8400, respectively. Furthermore, 53.7% of these markers could be transferred to the related species, Vicia faba. CONCLUSIONS: The developed markers have potential for application in the study of genetic diversity, germplasm appraisal, and marker-assisted breeding in pea and other legume species.

Soybean TCP transcription factors: Evolution, classification, protein interaction and stress and hormone responsiveness.

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, a family of plant-specific proteins, play crucial roles in plant growth and development and stress response. However, systematical information is unknown regarding the TCP gene family in soybean. In the present study, a total of 54 GmTCPs were identified in soybean, which were grouped into 11 groups with the typical TCP conserved domains. Phylogenetic relationship, protein motif and gene structure analyses distinguished the GmTCPs into two homology classes: Class I and Class II. Class II was then differentiated into two subclasses: CIN and CYC/TB1. Unique cis-element number and composition existed in the promoter regions which might be involved in the gene transcriptional regulation of different GmTCPs. Tissue expression analysis demonstrated the diverse spatiotemporal expression profiles of GmTCPs. Furthermore, the interaction protein of one previously functionally unknown TCP protein-GmTCP8 was investigated. Yeast two-hybrid assay showed the interaction between GmTCP8 and an abscisic acid receptor (GmPYL10). QRT-PCR assays indicated the distinct expression profiles of GmTCPs in response to abiotic stresses (heat, drought and salt) and stress-related signals (abscisic acid, brassinolide, salicylicacid and methyl jasmonate). These results will facilitate to uncover the possible roles of GmTCPs under abiotic stress and hormone signal responses in soybean.

Identification of the AQP members involved in abiotic stress responses from Arabidopsis.

Aquaporins (AQPs) constitute a highly diverse family of water channel proteins that play crucial biological functions in plant growth and development and stress physiology. In Arabidopsis, 35 AQPs are classified into four subfamilies (PIPs, TIPs, NIPs and SIPs). However, knowledge about the roles of different subfamily AQPs remains limited. Here, we explored the chromosomal location, gene structure and expression patterns of all AQPs in different tissues or under different abiotic stresses based on available microarray data. Tissue expression analysis showed that different AQPs had various expression patterns in tissues (root, leaf, flower and seed). Expression profiles under stress conditions revealed that most AQPs were responsive to osmotic, salt and drought stresses. Phenotypic and physiological identification showed that Tip2;2 loss-of-function mutant exhibited less sensitive to abiotic stresses (mannitol, NaCl and PEG) compared with wild-type, as evident by analysis of germination rate, root growth, survival rate, ion leakage, malondialdehyde (MDA) and proline contents. Mutant of TIP2;2 modulated the transcript levels of SOS1, SOS2, SOS3, DREB1A, DREB2A and P5CS1, under abiotic stress conditions. This study provides a basis for further functional identification of stress-related candidate AQPs in Arabidopsis.

Determination of the genetic diversity of vegetable soybean [Glycine max (L.) Merr.] using EST-SSR markers.

The development of expressed sequence tag-derived simple sequence repeats (EST-SSRs) provided a useful tool for investigating plant genetic diversity. In the present study, 22 polymorphic EST-SSRs from grain soybean were identified and used to assess the genetic diversity in 48 vegetable soybean accessions. Among the 22 EST-SSR loci, tri-nucleotides were the most abundant repeats, accounting for 50.00% of the total motifs. GAA was the most common motif among tri-nucleotide repeats, with a frequency of 18.18%. Polymorphic analysis identified a total of 71 alleles, with an average of 3.23 per locus. The polymorphism information content (PIC) values ranged from 0.144 to 0.630, with a mean of 0.386. Observed heterozygosity (Ho) values varied from 0.0196 to 1.0000, with an average of 0.6092, while the expected heterozygosity (He) values ranged from 0.1502 to 0.6840, with a mean value of 0.4616. Principal coordinate analysis and phylogenetic tree analysis indicated that the accessions could be assigned to different groups based to a large extent on their geographic distribution, and most accessions from China were clustered into the same groups. These results suggest that Chinese vegetable soybean accessions have a narrow genetic base. The results of this study indicate that EST-SSRs from grain soybean have high transferability to vegetable soybean, and that these new markers would be helpful in taxonomy, molecular breeding, and comparative mapping studies of vegetable soybean in the future.

Generation and characterization of 11 novel est derived microsatellites from Vicia faba (Fabaceae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) markers were developed for faba bean using expressed sequence tags (ESTs) from the NCBI database to study for genetic diversity. • METHODS AND RESULTS: A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe. The number of alleles (A) ranged from 1 to 3 in each population, and observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0 to 0.5000 and 0.6400, respectively. Furthermore, transferable analysis revealed that eight of these loci (72.73%) amplified in Pisum sativum L., six of which (75.00%) detected polymorphism. • CONCLUSIONS: The developed markers in this study will provide valuable tools for genetic diversity, resource conservation, genetic mapping, and marker-assisted breeding of faba bean in the future.

Developing new SSR markers from ESTs of pea (Pisum sativum L.).

The development of expressed sequence tags (ESTs) from pea has provided a useful source for mining novel simple sequence repeat (SSR) markers. In the present research, in order to find EST-derived SSR markers, 18 552 pea ESTs from the National Center for Biotechnology Information (NCBI) database were downloaded and assembled into 10 086 unigenes. A total of 586 microsatellites in 530 unigenes were identified, indicating that merely 5.25% of sequences contained SSRs. The most abundant SSRs within pea were tri-nucleotide repeat motifs, and among all the tri-nucleotide repeats, the motif GAA was the most abundant type. In total, 49 SSRs were used for primer design. EST-SSR loci were subsequently screened on 10 widely adapted varieties in China. Of these, nine loci showed polymorphic profiles that revealed two to three alleles per locus. The polymorphism information content value ranged from 0.18 to 0.58 with an average of 0.41. Furthermore, transferable analysis revealed that some of these loci showed transferability to faba bean. Because of their polymorphism and transferability, these nine novel EST-SSRs will be valuable tools for marker-assisted breeding and comparative mapping of pea in the future.

[Effects of NaCl stress on free polyamines content and reactive oxygen species level in pumpkin roots].

Taking Cucurbita moschata Duch hybrid 360-3 x 112-2 and C. ficifolia Bouche as test materials, the effects of NaCl stress on their plant growth and the O2*- production rate and H2O2 and free polyamines (PAs) contents in their roots were studied with hydroponic culture. The results showed that after 10 d NaCl stress, the plant growth of the two pumpkin varieties was strongly inhibited, compared with the control, and C. ficifolia was more injured than hybrid 360-3 x 112-2. Under NaCl stress, the root O2*- production rate and H2O2 content of the two pumpkin varieties were increased, but their absolute values were lower in hybrid 360-3 x 112-2 than in C. ficifolia. The contents of PAs, putrescine (Put), spermidine (Spd) and spermine (Spm) and Put/PAs ratio in the roots of the two pumpkin varieties were always higher than the control and had a trend of increased first and decreased then; while the (Spd + Spm)/Put ratio was lower than the control and decreased first and increased then. Compared with C. ficifolia Bouche, hybrid 360-3 x 112-2 always had a lower Put/PAs ratio and a lower Put content in its roots, but the (Spd + Spm)/Put ratio and Spd and Spm contents were always higher. It was concluded that under NaCl stress, the increasing PAs content in the roots of test materials played an active role in decreasing or scavenging reactive oxygen species (ROS). The conversion of Put to Spd and Spm was advantageous to the increase of plant salt tolerance. The higher salt tolerance of hybrid 360-3 x 112-2 was closely related to the lower Put/PAs ratio and the higher (Spd + Spm)/Put ratio and PAs content in its roots, and thus, the stronger capacity to scavenge ROS.

[Effects of NaCl stress on the growth, antioxidant enzyme activities and reactive oxygen metabolism of grafted eggplant].

By the method of hydroponics and with the salt-tolerant eggplant cultivar 'Torvum Vigor' (Solanum torvum) from Japan as rootstock and the cultivar 'Suqiqie' (Solanum melongena L. ) as scion, this paper studied the differences between grafted and own-root seedlings in their photosynthetic characteristics, antioxidant enzyme activities, and reactive oxygen metabolism under 80 mmol x L(-1) NaCl stress. The results showed that under NaCl stress, the dry mass, chlorophyll content and photosynthetic rate of grafted seedlings were 67.8%, 8. 8% and 31.1% higher than those of own-root seedlings, respectively, and the antioxidant enzyme activities were significantly higher while the O2* producing rate and the hydrogen peroxide (H2O2) and malondiadehyde (MDA) contents were significantly lower in grafted seedlings leaves than in own-root seedlings. For both grafted and own-root seedlings, NaCl stress reduced the chlorophyll content and photosynthetic rate, but increased the O2* producing rate and the H2O2 and MDA contents significantly. However, the growth of grafted seedlings was less affected by NaCl stress. The stronger salt tolerance of grafted eggplant seedlings was related to their higher antioxidant enzyme activities and less oxidative damage.

[Effects of NaCl stress on polyamine contents in grafted cucumber leaves].

With a Japanese salt-tolerant pumpkin cultivar 'King Shintosa' (Cucurbita maxima x C. moschata) as rootstock and a cucumber (Cucumis sativus) cultivar 'Xintaimici' as cion, this paper studied the temporal dynamics of different form polyamines contents in the leaves of grafted and own-rooted cucumber plants under 100 mmol x L(-1) NaCl stress. The results showed that the free putrescence (Put) content of graftedplant leaves (G2) was significantly higher than that of own-rooted plant leaves (O2), except on the second day of NaCl stress. During the whole period of NaCl stress, the free spermidine (Spd) and spermine (Spm) contents of G2 were significantly higher than those of O2, and the total free polyamine content of G2 peaked on the fourth day of stress. The free Put/PAs value of G2 was significantly lower than that of O2 except on the fourth day of stress, but (Spd + Spm)/Put value was significantly higher than that of O2 during the whole period of stress. In the test period, the contents of conjugated and bound Put, Spd, Spm of G2 were significantly higher than those of O2. The total conjugated and bound polyamine contents of G2 peaked on the sixth day of stress. Both conjugated Put/PAs and conjugated (Spd + Spm)/Put values had a similar changing trend to free polyamine. The bound Put/PAs value of G2 was significantly lower than that of O2 except on the sixth day of stress, while the bound (Spd + Spm)/Put value was significantly higher than that of O2 during the whole period of stress. All of these demonstrated that grafted cucumber plant possessed a stronger salt tolerance.