SUnSeT: spectral unmixing of hyperspectral images for phenotyping soybean seed traits.

KEY MESSAGE: Hyperspectral features enable accurate classification of soybean seeds using linear discriminant analysis and GWAS for novel seed trait genes. Evaluating crop seed traits such as size, shape, and color is crucial for assessing seed quality and improving agricultural productivity. The introduction of the SUnSet toolbox, which employs hyperspectral sensor-derived image analysis, addresses this necessity. In a validation test involving 420 seed accessions from the Korean Soybean Core Collections, the pixel purity index algorithm identified seed- specific hyperspectral endmembers to facilitate segmentation. Various metrics extracted from ventral and lateral side images facilitated the categorization of seeds into three size groups and four shape groups. Additionally, quantitative RGB triplets representing seven seed coat colors, averaged reflectance spectra, and pigment indices were acquired. Machine learning models, trained on a dataset comprising 420 accession seeds and 199 predictors encompassing seed size, shape, and reflectance spectra, achieved accuracy rates of 95.8% for linear discriminant analysis model. Furthermore, a genome-wide association study utilizing hyperspectral features uncovered associations between seed traits and genes governing seed pigmentation and shapes. This comprehensive approach underscores the effectiveness of SUnSet in advancing precision agriculture through meticulous seed trait analysis.

Peptide transporter2 (PTR2) enhances water uptake during early seed germination in Arabidopsis thaliana.

KEY MESSAGE: PTR2 in Arabidopsis thaliana is negatively regulated by ABI4 and plays a key role in water uptake by seeds, ensuring that imbibed seeds proceed to germination. Peptide transporters (PTRs) transport nitrogen-containing substrates in a proton-dependent manner. Among the six PTRs in Arabidopsis thaliana, the physiological role of the tonoplast-localized, seed embryo abundant PTR2 is unknown. In the present study, a molecular physiological analysis of PTR2 was conducted using ptr2 mutants and PTR2CO complementation lines. Compared with the wild type, the ptr2 mutant showed ca. 6 h delay in testa rupture and consequently endosperm rupture because of 17% lower water content and 10% higher free abscisic acid (ABA) content. Constitutive overexpression of the PTR2 gene under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in ptr2 mutants rescued the mutant phenotypes. After cold stratification, a transient increase in ABA INSENSITIVE4 (ABI4) transcript levels during induction of testa rupture was followed by a similar increase in PTR2 transcript levels, which peaked prior to endosperm rupture. The PTR2 promoter region containing multiple CCAC motifs was recognized by ABI4 in electrophoretic mobility shift assays, and PTR2 expression was repressed by 67% in ABI4 overexpression lines compared with the wild type, suggesting that PTR2 is an immediate downstream target of ABI4. Taken together, the results suggest that ABI4-dependent temporal regulation of PTR2 expression may influence water status during seed germination to promote the post-germinative growth of imbibed seeds.

A wheat R2R3-MYB protein PURPLE PLANT1 (TaPL1) functions as a positive regulator of anthocyanin biosynthesis.

Transcriptional activation of anthocyanin biosynthesis genes in vegetative tissues of monocotyledonous plants is mediated by cooperative activity of one component from each of the following two transcription factor families: MYB encoded by PURPLE PLANT1/COLORED ALEURONE1 (PL1/C1), and basic helix-loop-helix (bHLH) encoded by RED/BOOSTER (R1/B1). In the present study, putative PL cDNA was cloned from the wheat (Triticum aestivum) cultivar Iksan370, which preferentially expresses anthocyanins in coleoptiles. Phylogenetic tree analysis of deduced amino acid sequences showed that a putative TaPL1 is highly homologous to barley (Hordeum vulgare) HvPL1, but is distinct from wheat TaC1. Transgenic Arabidopsis thaliana stably expressing putative TaPL1 accumulated anthocyanin pigments in leaves and up-regulated structural genes involved in both early and late anthocyanin biosynthesis steps. TaPL1 transcript levels in Iksan370 were more prominent in vegetative tissues such as young coleoptiles than in reproductive tissues such as spikelets. TaPL1 expression was significantly up-regulated by environmental stresses including cold, salt, and light, which are known to induce anthocyanin accumulation. These combined results suggest that TaPL1 is an active positive regulator of anthocyanin biosynthesis in wheat coleoptiles.

Identification of genes that may regulate the expression of the transcription factor production of anthocyanin pigment 1 (PAP1)/MYB75 involved in Arabidopsis anthocyanin biosynthesis.

KEY MESSAGE: A putative RNA-binding protein with a single RNA Recognition Motif (At3G63450) is involved in anthocyanin biosynthesis via its ability to modulate the transcript level of a major positive regulator PAP1 in Arabidopsis. The R2R3 MYB-activator production of anthocyanin pigment 1 (PAP1)/MYB75 plays a major role in anthocyanin biosynthesis in Arabidopsis in combination with one of three bHLH activators including transparent test 8 (TT8), enhancer of glabra3 (EGL3), glabra3 (GL3), and the WD-repeat transcription factor transparent testa 1 (TTG1), forming ternary MYB-basic HLH-WD40 complexes. Transcriptional activation of PAP1 expression is largely triggered by changes in light color and intensity, temperature fluctuations, nutrient status, and sugar and hormone treatments. However, the immediate upstream and downstream regulatory factors for PAP1 transcription are largely unknown. In the present study, using a T-DNA insertional mutagenesis approach, we transformed pap1-Dominant (pap1D) plants to modulate the levels of endogenous PAP1 transcripts. We employed Restriction Site Extension (RSE)-PCR analysis of 247 homogenous T3 genetic mutant lines exhibiting variations in anthocyanin accumulation compared to pap1D and identified 92 lines with T-DNA integrated in either intra- or inter-genic locations. This analysis revealed 80 novel candidate proteins, including a putative RNA-binding protein with a single RNA Recognition Motif (At3G63450), which may directly or indirectly regulate PAP1 expression at the transcriptional level.

Calcium dependent sucrose uptake links sugar signaling to anthocyanin biosynthesis in Arabidopsis.

Sugars enhance light signaling-induced anthocyanin accumulation in Arabidopsis seedlings via differential regulation of several positive and negative transcription factors. Ca(2+) plays a role as a second messenger in sugar signaling in grape and wheat. However, whether anthocyanin pigmentation is modulated by changes in intracellular Ca(2+) level in Arabidopsis is not known. Here, we used a pharmaceutical approach that Ca(2+) antagonists strongly interfered with sucrose uptake and anthocyanin accumulation by downregulating the expression of sucrose transporter 1 (SUC1) and transcriptional regulatory factors, such as PAP1. Time course analysis of the effect of Ca(2+) antagonists showed the early inhibition of sucrose-induced sugar uptake leading to decreased anthocyanin accumulation, indicating that Ca(2+) signals play a role in sugar uptake rather than in anthocyanin biosynthesis. An early increase in cytosolic Ca(2+) level in Arabidopsis roots in response to sucrose feeding was significantly inhibited by Ca(2+) antagonists. Taken together, these results indicate that sucrose-induced sugar uptake in Arabidopsis is modulated by changes in endogenous Ca(2+) levels, which in turn regulate anthocyanin accumulation.

Modulation of warm temperature-sensitive growth using a phytochrome B dark reversion variant, phyB[G515E], in Arabidopsis and rice.

INTRODUCTION: Ambient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB-PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. OBJECTIVES: This study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. METHODS: The thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. RESULTS: At an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. CONCLUSION: Transient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4-auxin-mediated thermomorphogenic responses in plants.