Effect of lead on physiological and antioxidant responses in two Vigna unguiculata cultivars differing in Pb-accumulation.

Lead (Pb) is one of the most toxic anthropogenic pollutants, occurring widely in both terrestrial and aquatic ecosystems, where it impairs plant growth and development. In this work, the effect of 0.5 mM EDTA-Pb was evaluated in two Vigna unguiculata cultivars (SV and SET), with the aim of detecting genotype/cultivar dependent changes in the physiological and anti-oxidant responses (CAT and APX) of a leguminous plant. The data showed that SV accumulated more Pb in roots while SET accumulated more in leaves, indicating differential regulation in Pb-translocation/accumulation. Lead affected the growth of SV less severely than SET, mainly associated with reduced inhibition in photosynthetic parameters. Furthermore, CAT and APX activities increased or were sustained at elevated levels in both cultivars in response to lead. However, gene expression analyses revealed that CAT1 was the main lead responsive gene in SET while CAT2 was more responsive in SV. APX1 was higher expressed in tissues with higher Pb-accumulation while APX2 was ubiquitously responsive to lead in both cultivars. Taken together, these results reveal differential ability of V. unguiculata cultivars in Pb-accumulation in different tissues affecting distinctly physiological and anti-oxidant responses. In addition, the existence of cultivars with predominant Pb-accumulation in aerial tissues invokes a need for studies to identify pollution-safe cultivars of leguminous plants to ensure food safety.

Phylogenetic analysis and differential expression of EF1α genes in soybean during development, stress and phytohormone treatments.

The EF1α is a multifunctional protein with additional unrelated activities to its primary function in translation. This protein is encoded by a multigene family and few studies are still available in plants. Expression of six EF1α genes in Glycine max was performed using RT-qPCR and RNA-seq data to advance in the function of each gene during plant development, stress conditions and phytohormone treatments. A phylogenetic classification in Phaseoleae tribe was used to identify the G. max EF1α genes (EF1α 1a1, 1a2, 1b, 2a, 2b and 3). Three EF1α types (1-3) were found in Phaseoleae revealing duplications in G. max types 1 and 2. EF1α genes were expressed in all studied tissues, however, specific amount of each transcript was detected. In plant development, all EF1α transcripts were generally more expressed in younger tissues, however, in unifoliolate leaves and cotyledons a higher expression occurred in older tissues. Five EF1α genes (except 2a) were up-regulated under stress in a response tissue/stress/cultivar-dependent. EF1α 3 was the most stress-induced gene linked to cultivar stress tolerance mainly in aerial tissues. Auxin, salicylate and ethylene induced differentially the EF1α expression. Overall, this study provides a consistent EF1α classification in Phaseoleae tribe to better understand their functional evolution. The RT-qPCR and RNA-seq EF1α expression profiles were consistent, both exhibiting expression diversification of each gene (spatio-temporal, stress and phytohormone stimuli). Our results point out the EF1α genes, especially EF1α 3, as candidate for developing a useful tool for future G. max breeding.

Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions.

KEY MESSAGE: The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1ß and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1ß), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

A classification scheme for alternative oxidases reveals the taxonomic distribution and evolutionary history of the enzyme in angiosperms.

A classification scheme based on protein phylogenies and sequence harmony method was used to clarify the taxonomic distribution and evolutionary history of the alternative oxidase (AOX) in angiosperms. A large data set analyses showed that AOX1 and AOX2 subfamilies were distributed into 4 phylogenetic clades: AOX1a-c/1e, AOX1d, AOX2a-c and AOX2d. High diversity in AOX family compositions was found. While the AOX2 subfamily was not detected in monocots, the AOX1 subfamily has expanded (AOX1a-e) in the large majority of these plants. In addition, Poales AOX1b and 1d were orthologous to eudicots AOX1d and then renamed as AOX1d1 and 1d2. AOX1 or AOX2 losses were detected in some eudicot plants. Several AOX2 duplications (AOX2a-c) were identified in eudicot species, mainly in the asterids. The AOX2b originally identified in eudicots in the Fabales order (soybean, cowpea) was divergent from AOX2a-c showing some specific amino acids with AOX1d and then it was renamed as AOX2d. AOX1d and AOX2d seem to be stress-responsive, facultative and mutually exclusive among species suggesting a complementary role with an AOX1(a) in stress conditions. Based on the data collected, we present a model for the evolutionary history of AOX in angiosperms and highlight specific areas where further research would be most beneficial.

Stress-induced co-expression of two alternative oxidase (VuAox1 and 2b) genes in Vigna unguiculata.

Cowpea (Vigna unguiculata) alternative oxidase is encoded by a small multigene family (Aox1, 2a and 2b) that is orthologous to the soybean Aox family. Like most of the identified Aox genes in plants, VuAox1 and VuAox2 consist of 4 exons interrupted by 3 introns. Alignment of the orthologous Aox genes revealed high identity of exons and intron variability, which is more prevalent in Aox1. In order to determine Aox gene expression in V. unguiculata, a steady-state analysis of transcripts involved in seed development (flowers, pods and dry seeds) and germination (soaked seeds) was performed and systemic co-expression of VuAox1 and VuAox2b was observed during germination. The analysis of Aox transcripts in leaves from seedlings under different stress conditions (cold, PEG, salicylate and H2O2 revealed stress-induced co-expression of both VuAox genes. Transcripts of VuAox2a and 2b were detected in all control seedlings, which was not the case for VuAox1 mRNA. Estimation of the primary transcript lengths of V. unguiculata and soybean Aox genes showed an intron length reduction for VuAox1 and 2b, suggesting that the two genes have converged in transcribed sequence length. Indeed, a bioinformatics analysis of VuAox1 and 2b promoters revealed a conserved region related to a cis-element that is responsive to oxidative stress. Taken together, the data provide evidence for co-expression of Aox1 and Aox2b in response to stress and also during the early phase of seed germination. The dual nature of VuAox2b expression (constitutive and induced) suggests that the constitutive Aox2b gene of V. unguiculata has acquired inducible regulatory elements.

Alternative oxidase regulation in roots of Vigna unguiculata cultivars differing in drought/salt tolerance.

The alternative oxidase (Aox) was studied at different levels (transcript, protein and capacity) in response to an osmotic shock applied to roots of cowpea (Vigna unguiculata). Two cultivars of V. unguiculata were used, Vita 3 and Vita 5, tolerant and sensitive to drought/saline stress respectively. The seedlings (17-day-old) were grown in hydroponic conditions and submitted to NaCl (100 and 200 mM) or 200.67 g L(-1) PEG 6000 (iso-osmotic condition to 100 mM NaCl). The VuAox1 and VuAox2a mRNA were not detected in either cultivar under all tested conditions while the VuAox2b gene was differently expressed. In the tolerant cultivar (Vita 3), the expression of VuAox2b gene was stimulated by an osmotic stress induced by PEG which was associated with a higher amount and capacity of the Aox protein. In the same cultivar, this gene was under-expressed in salt stress conditions with poor effect on the protein level. In the sensitive cultivar (Vita 5), the transcript level of the VuAox2b was unchanged in response to PEG treatment, even though the protein and the capacity tended to increase. Upon salt stress, the VuAox2b gene was over-expressed. At 100mM NaCl, this VuAox2b gene over-expression led to a higher amount and capacity of Aox. This effect was reduced at 200 mM NaCl. Overall, these results suggest complex mechanisms (transcriptional, translational and post-translational) for Aox regulation in response to osmotic stress.