It is well established that 14-3-3 proteins are key regulators of multiple stress signal transduction cascades. However, the biological functions of soybean 14-3-3 proteins, especially in plant drought response, are not yet known. In this study, we characterized a Glycine soja 14-3-3 gene, GsGF14o, which is involved in plant development and drought response. GsGF14o expression was greatly induced by drought stress, as evidenced by the quantitative real-time PCR and β-glucuronidase (GUS) activity analysis. GsGF14o overexpression in Arabidopsis thaliana resulted in decreased drought tolerance during seed germination and seedling growth. Furthermore, silencing of AtGF14µ, the most homologous 14-3-3 gene of GsGF14o, led to enhanced drought tolerance at both the seed germination and seedling stage. Unexpectedly, GsGF14o transgenic lines showed reduced water loss and transpiration rates compared with wild-type plants, which was demonstrated to be the consequence of the decreased stomatal size. At the same time, the smaller stomata due to GsGF14o overexpression led to a relatively slow net photosynthesis rate, which led to a growth penalty under drought stress. We further demonstrated that GsGF14o overexpression caused deficits in root hair formation and development, and thereby reduced the water intake capacity of the transgenic root system. In addition, GsGF14o overexpression down-regulated the transcript levels of drought-responsive marker genes. Finally, we also investigated the tissue-specific accumulation of GsGF14o by using a GUS activity assay. Collectively, the results presented here confirm that GsGF14o plays a dual role in drought stress responses through its involvement in the regulation of stomatal size and root hair development.

It has been well demonstrated that cystatins regulated plant stress tolerance through inhibiting the cysteine proteinase activity under environmental stress. However, there was limited information about the role of cystatins in plant alkali stress response, especially in wild soybean. Here, in this study, we focused on the biological characterization of a novel Glycine soja cystatin protein GsCPI14, which interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and positively regulated plant alkali stress tolerance. The protein-protein interaction between GsCBRLK and GsCPI14 was confirmed by using split-ubiquitin based membrane yeast two-hybrid analysis and bimolecular fluorescence complementation assay. Expression of GsCPI14 was greatly induced by salt, ABA and alkali stress in G. soja, and GsCBRLK overexpression (OX) in Glycine max promoted the stress induction of GmCPI14 expression under stress conditions. Furthermore, we found that GsCPI14-eGFP fusion protein localized in the entire Arabidopsis protoplast and onion epidermal cell, and GsCPI14 showed ubiquitous expression in different tissues of G. soja. In addition, we gave evidence that the GST-GsCPI14 fusion protein inhibited the proteolytic activity of papain in vitro. At last, we demonstrated that OX of GsCPI14 in Arabidopsis promoted the seed germination under alkali stress, as evidenced by higher germination rates. GsCPI14 transgenic Arabidopsis seedlings also displayed better growth performance and physiological index under alkali stress. Taken together, results presented in this study demonstrated that the G. soja cysteine proteinase inhibitor GsCPI14 interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and regulated plant tolerance to alkali stress.

Spatial interpolation method is the basis of soil heavy metal pollution assessment and remediation. The existing evaluation index for interpolation accuracy did not combine with actual situation. The selection of interpolation methods needs to be based on specific research purposes and research object characteristics. In this paper, As pollution in soils of Beijing was taken as an example. The prediction accuracy of ordinary kriging (OK) and inverse distance weighted (IDW) were evaluated based on the cross validation results and spatial distribution characteristics of influencing factors. The results showed that, under the condition of specific spatial correlation, the cross validation results of OK and IDW for every soil point and the prediction accuracy of spatial distribution trend are similar. But the prediction accuracy of OK for the maximum and minimum is less than IDW, while the number of high pollution areas identified by OK are less than IDW. It is difficult to identify the high pollution areas fully by OK, which shows that the smoothing effect of OK is obvious. In addition, with increasing of the spatial correlation of As concentration, the cross validation error of OK and IDW decreases, and the high pollution area identified by OK is approaching the result of IDW, which can identify the high pollution areas more comprehensively. However, because the semivariogram constructed by OK interpolation method is more subjective and requires larger number of soil samples, IDW is more suitable for spatial prediction of heavy metal pollution in soils.

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Heterodera avenae is one of the most important plant pathogens and causes vast losses in cereal crops. As a sedentary endoparasitic nematode, H. avenae secretes effectors that modify plant defenses and promote its biotrophic infection of its hosts. However, the number of effectors involved in the interaction between H. avenae and host defenses remains unclear. Here, we report the identification of putative effectors in H. avenae that regulate plant defenses on a large scale. Our results showed that 78 of the 95 putative effectors suppressed programmed cell death (PCD) triggered by BAX and that 7 of the putative effectors themselves caused cell death in Nicotiana benthamiana. Among the cell-death-inducing effectors, three were found to be dependent on their specific domains to trigger cell death and to be expressed in esophageal gland cells by in situ hybridization. Ten candidate effectors that suppressed BAX-triggered PCD also suppressed PCD triggered by the elicitor PsojNIP and at least one R-protein/cognate effector pair, suggesting that they are active in suppressing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Notably, with the exception of isotig16060, these putative effectors could also suppress PCD triggered by cell-death-inducing effectors from H. avenae, indicating that those effectors may cooperate to promote nematode parasitism. Collectively, our results indicate that the majority of the tested effectors of H. avenae may play important roles in suppressing cell death induced by different elicitors in N. benthamiana.

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Plant parasitic nematodes secrete effectors into host plant tissues to facilitate parasitism. In this study, we identified a G16B09-like effector protein family from the transcriptome of Heterodera avenae, and then verified that most of the members could suppress programmed cell death triggered by BAX in Nicotiana benthamiana. Ha18764, the most homologous to G16B09, was further characterized for its function. Our experimental evidence suggested that Ha18764 was specifically expressed in the dorsal gland and was dramatically upregulated in the J4 stage of nematode development. A Magnaporthe oryzae secretion system in barley showed that the signal peptide of Ha18764 had secretion activity to deliver mCherry into plant cells. Arabidopsis thaliana overexpressing Ha18764 or Hs18764 was more susceptible to Heterodera schachtii. In contrast, BSMV-based host-induced gene silencing (HIGS) targeting Ha18764 attenuated H. avenae parasitism and its reproduction in wheat plants. Transient expression of Ha18764 suppressed PsojNIP, Avr3a/R3a, RBP-1/Gpa2, and MAPK kinases (MKK1 and NPK1Nt)-related cell death in Nicotiana benthamiana. Co-expression assays indicated that Ha18764 also suppressed cell death triggered by four H. avenae putative cell-death-inducing effectors. Moreover, Ha18764 was also shown strong PTI suppression such as reducing the expression of plant defense-related genes, the burst of reactive oxygen species, and the deposition of cell wall callose. Together, our results indicate that Ha18764 promotes parasitism, probably by suppressing plant PTI and ETI signaling in the parasitic stages of H. avenae.