Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.

The Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV) that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W) residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S). The mutated OsARF(WS)s can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WS)s in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs

Stability Analysis and Measure Study of Tobacco Farmers in the Mountainous Area of Western Hubei

Through the investigation, this paper analyzes the important restricting factors for the tobacco farmers in Enshi Badong mountainous area from 2018 to 2021, including the age structure, the loss situation, the number of years of planting tobacco, the scale of planting tobacco and the benefit of planting tobacco. This paper puts forward some corresponding measures and suggestions, such as cultivating the team of professional tobacco farmers, establishing the team of young tobacco farmers, expanding professional services, developing "tobacco plus" diversified industries, and popularizing suitable agricultural machinery in mountainous areas in order to effectively improve the stability of the team of tobacco farmers

HA-Unet: A Modified Unet Based on Hybrid Attention for Urban Water Extraction in SAR Images

Urban water plays a significant role in the urban ecosystem, but urban water extraction is still a challenging task in automatic interpretation of synthetic aperture radar (SAR) images. The influence of radar shadows and strong scatters in urban areas may lead to misclassification in urban water extraction. Nevertheless, the local features captured by convolutional layers in Convolutional Neural Networks (CNNs) are generally redundant and cannot make effective use of global information to guide the prediction of water pixels. To effectively emphasize the identifiable water characteristics and fully exploit the global information of SAR images, a modified Unet based on hybrid attention mechanism is proposed to improve the performance of urban water extraction in this paper. Considering the feature extraction ability and the global modeling capability in SAR image segmentation, the Channel and Spatial Attention Module (CSAM) and the Multi-head Self-Attention Block (MSAB) are both introduced into the proposed Hybrid Attention Unet (HA-Unet). In this work, Resnet50 is adopted as the backbone of HA-Unet to extract multi-level features of SAR images. During the feature extraction process, CSAM based on local attention is adopted to enhance the meaningful water features and ignore unnecessary features adaptively in feature maps of two shallow layers. In the last two layers of the backbone, MSAB is introduced to capture the global information of SAR images to generate global attention. In addition, two global attention maps generated by MSAB are aggregated together to reconstruct the spatial feature relationship of SAR images from high-resolution feature maps. The experimental results on Sentinel-1A SAR images show that the proposed urban water extraction method has a strong ability to extract water bodies in the complex urban areas. The ablation experiment and visualization results vividly indicate that both CSAM and MSAB contribute significantly to extracting urban water accurately and effectively

HA-Unet: A Modified Unet Based on Hybrid Attention for Urban Water Extraction in SAR Images

Urban water plays a significant role in the urban ecosystem, but urban water extraction is still a challenging task in automatic interpretation of synthetic aperture radar (SAR) images. The influence of radar shadows and strong scatters in urban areas may lead to misclassification in urban water extraction. Nevertheless, the local features captured by convolutional layers in Convolutional Neural Networks (CNNs) are generally redundant and cannot make effective use of global information to guide the prediction of water pixels. To effectively emphasize the identifiable water characteristics and fully exploit the global information of SAR images, a modified Unet based on hybrid attention mechanism is proposed to improve the performance of urban water extraction in this paper. Considering the feature extraction ability and the global modeling capability in SAR image segmentation, the Channel and Spatial Attention Module (CSAM) and the Multi-head Self-Attention Block (MSAB) are both introduced into the proposed Hybrid Attention Unet (HA-Unet). In this work, Resnet50 is adopted as the backbone of HA-Unet to extract multi-level features of SAR images. During the feature extraction process, CSAM based on local attention is adopted to enhance the meaningful water features and ignore unnecessary features adaptively in feature maps of two shallow layers. In the last two layers of the backbone, MSAB is introduced to capture the global information of SAR images to generate global attention. In addition, two global attention maps generated by MSAB are aggregated together to reconstruct the spatial feature relationship of SAR images from high-resolution feature maps. The experimental results on Sentinel-1A SAR images show that the proposed urban water extraction method has a strong ability to extract water bodies in the complex urban areas. The ablation experiment and visualization results vividly indicate that both CSAM and MSAB contribute significantly to extracting urban water accurately and effectively

The W residue in Domain IV of OsARFs is crucial for the protein-protein interactions between Osiaa23 and OsARFs.

<p>(A) Diagram of conserved domains of OsARF protein. Black rectangles indicate four domains of OsARF, DBD and MR represent DNA binding Domain and middle region respectively, III and IV represent Domain III and Domain IV, which are similar to that of OsIAA. The substitution from W to S in Domain IV is indicated by arrow. (B) Self-activation test of OsARF16 and OsARF16(WS), which has an amino acid substitution from W to S in Domain IV. The transformed yeast was grown on medium without tryptophan, histidine and ade (SD -Trp/ -His/ -Ade). 1, OsARF16; 2, OsARF16(WS); 3, negative control. (C-D) Interactions between Osiaa23 and OsARFs, Osiaa23 and OsARF(WS)s in the yeast two-hybrid system. 1, positive control; 2, negative control; 3, Osiaa23 + OsARF6; 4, Osiaa23 + OsARF12; 5, Osiss23 + OsARF16; 6, Osiaa23 + OsARF17; 7, Osiaa23 + OsARF25; 8, Osiaa23 + OsARF6(WS); 9, Osiaa23 + OsARF12(WS); 10, Osiaa23 + OsARF16(WS); 11, Osiaa23 + OsARF17(WS); 12, Osiaa23 + OsARF25(WS). Yeast was grown on medium without leucine and tryptophan (SD –Leu/ -Trp) as a contral (C) and medium without leucine, tryptophan, histidine and ade (SD -Trp/ -Leu/ -Ade/ -His) to test the protein-protein interactions (D).</p

The W residue in Domain IV is conserved in both OsIAAs and OsARFs.

<p>(A) The alignment of Domain IV of OsAux/IAAs in rice, the conserved W is marked by the arrow. Conserved GDVP motif is indicated by thick line above the alignment. (B) The alignment of Domain IV of OsARFs in rice, the conserved W is marked by the arrow. Conserved GDDP motif is indicated by thick line above the alignment.</p

Amino acid substitution in Domain IV of Osiaa23 prevents the protein-protein interactions between Osiaa23 and OsARFs.

<p>Interactions between Osiaa23 and OsARFs, Osiaa23-R5 and OsARFs in the yeast two-hybrid system. 1, positive control; 2, negative control; 3, Osiaa23 + OsARF6; 4, Osiaa23 + OsARF12; 5, Osiss23 + OsARF16; 6, Osiaa23 + OsARF17; 7, Osiaa23 + OsARF25; 8, Osiaa23-R5 + OsARF6; 9, Osiaa23-R5 + OsARF12; 10, Osiaa23-R5 + OsARF16; 11, Osiaa23-R5 + OsARF17; 12, Osiaa23-R5 + OsARF25. Yeast was grown on medium without leucine and tryptophan (SD -Leu/ -Trp) as a contral (A) and medium without leucine, tryptophan, histidine and ade (SD -Trp/ -Leu/ -Ade/ -His) to test the protein-protein interactions (B).</p

Intragenic mutation in Domain IV fully rescued the defects of <i>Osiaa23-3</i> mutant.

<p>(A) Phenotypes of 7-day-old seedlings of wild type (WT), heterozygous mutant of <i>Osiaa23-3</i> (Aa), homozygous mutant of <i>Osiaa23-3</i> (aa) and the suppressor of <i>Osiaa23-3</i>, <i>Osiaa23-R5</i>. Bar  = 2 cm. (B) Diagram of conserved domains of OsIAA23. Black rectangles marked by Roman numerals indicate four domains of OsIAA23, and two mutated sites in Domain II and Domain IV are marked by arrows. (C) Growth parameters of 7-day-old seedlings of WT, Aa, aa and <i>Osiaa23-R5</i>. (D-G) Root tips of 7-day-old seedlings of WT (D), Aa (E), aa (F) and <i>Osiaa23-R5</i> (G). Bars  = 250 µm.</p

Mutated OsARF(WS)s rescued different defects of <i>Osiaa23-3</i>.

<p>The phenotypes of transgenic rice, over expressing <i>OsARF(WS)s</i> in the <i>Osiaa23-3</i> background. Two independent lines of transgenic rice over expressing <i>OsARF(WS)s</i> are compared with WT and <i>Osiaa23-3</i> in the aspects of shoot length (A), root length (B) and crown root number (C). Statistically distinct groups are marked by a, b and c (n = 10).</p