Transcriptional Landscape and Dynamics Involved in Sugar and Acid Accumulation during Apple Fruit Development.

In fleshy fruit, sugars and acids are central components of fruit flavor and quality. To date, the mechanisms underlying transcriptional regulation of sugar and acid during fruit development remain largely unknown. Here, we combined ATAC-seq with RNA-seq to investigate the genome-wide chromatin accessibility and to identify putative transcription factors related to sugar and acid accumulation during apple (Malus domestica) fruit development. By integrating the differentially accessible regions (DARs) and differentially expressed genes (DEGs), we generated a global dataset of promoter-accessibility- and expression-increased genes (PEIGs). Using this strategy, we constructed a transcriptional regulatory network enabling screening for key transcription factors and target genes involved in sugar and acid accumulation. Among these transcription factors, five fruit-specific Dof (DNA binding with one finger) genes were selected to confirm their regulatory effects, and our results showed that they could affect sugar or acid concentration by regulating the expression of sugar or acid metabolism-related genes in apple fruits. Our transcriptional regulatory network provides a suitable platform to identify candidate genes that control sugar and acid accumulation. Meanwhile, our dataset will aid in analyzing other characteristics of apple fruit that have not been illuminated previously. Overall, these findings support a better understanding of the regulatory dynamics during apple fruit development and lay a foundation for quality improvement of apple.

Plant elicitor Peptides regulate root hair development in Arabidopsis.

Plant Elicitor Peptides (Peps) induce plant immune responses and inhibit root growth through their receptors PEPR1 and PEPR2, two receptor-like kinases. In our study, we found a previously unknown function of Peps that enhance root hair growth in a PEPRs-independent manner. When we characterized the expression patterns of PROPEP genes, we found several gene promoters of PROPEP gene family were particularly active in root hairs. Furthermore, we observed that PROPEP2 is vital for root hair development, as disruption of PROPEP2 gene led to a significant reduction in root hair density and length. We also discovered that PROPEP2 regulates root hair formation via the modulation of CPC and GL2 expression, thereby influencing the cell-fate determination of root hairs. Additionally, calcium signaling appeared to be involved in PROPEP2/Pep2-induced root hair growth. These findings shed light on the function of Peps in root hair development.

The ABI4-RGL2 module serves as a double agent to mediate the antagonistic crosstalk between ABA and GA signals.

Abscisic acid (ABA) and gibberellins (GA) antagonistically mediate several biological processes, including seed germination, but the molecular mechanisms underlying ABA/GA antagonism need further investigation, particularly any role mediated by a transcription factors module. Here, we report that the DELLA protein RGL2, a repressor of GA signaling, specifically interacts with ABI4, an ABA signaling enhancer, to act as a transcription factor complex to mediate ABA/GA antagonism. The rgl2, abi3, abi4 and abi5 mutants rescue the non-germination phenotype of the ga1-t. Further, we demonstrate that RGL2 specifically interacts with ABI4 to form a heterodimer. RGL2 and ABI4 stabilize one another, and GA increases the ABI4-RGL2 module turnover, whereas ABA decreases it. At the transcriptional level, ABI4 enhances the RGL2 expression by directly binding to its promoter via the CCAC cis-element, and RGL2 significantly upregulates the transcriptional activation ability of ABI4 toward its target genes, including ABI5 and RGL2. Abscisic acid promotes whereas GA inhibits the ability of ABI4-RGL2 module to activate transcription, and ultimately ABA and GA antagonize each other. Genetic analysis demonstrated that both ABI4 and RGL2 are essential for the activity of this transcription factor module. These results suggest that the ABI4-RGL2 module mediates ABA/GA antagonism by functioning as a double agent.

Immunophilin FKB20-2 participates in oligomerization of Photosystem I in Chlamydomonas.

PSI is a sophisticated photosynthesis protein complex that fuels the light reaction of photosynthesis in algae and vascular plants. While the structure and function of PSI have been studied extensively, the dynamic regulation on PSI oligomerization and high light response is less understood. In this work, we characterized a high light-responsive immunophilin gene FKB20-2 (FK506-binding protein 20-2) required for PSI oligomerization and high light tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Biochemical assays and 77-K fluorescence measurement showed that loss of FKB20-2 led to the reduced accumulation of PSI core subunits and abnormal oligomerization of PSI complexes and, particularly, reduced PSI intermediate complexes in fkb20-2. It is noteworthy that the abnormal PSI oligomerization was observed in fkb20-2 even under dark and dim light growth conditions. Coimmunoprecipitation, MS, and yeast 2-hybrid assay revealed that FKB20-2 directly interacted with the low molecular weight PSI subunit PsaG, which might be involved in the dynamic regulation of PSI-light-harvesting complex I supercomplexes. Moreover, abnormal PSI oligomerization caused accelerated photodamage to PSII in fkb20-2 under high light stress. Together, we demonstrated that immunophilin FKB20-2 affects PSI oligomerization probably by interacting with PsaG and plays pivotal roles during Chlamydomonas tolerance to high light.

Accumulation of livestock manure-derived heavy metals in the Hexi Corridor oasis agricultural alkaline soil and bioavailability to Chinese cabbage (Brassica pekinensis L.) after 4-year continuous application.

Hexi Corridor is one of the most important base of vegetable producing areas in China. Livestock manure (LM) applied to agricultural field could lead to soil heavy metal (HM) pollution. Previous studies have focused on HM pollution following LM application in acidic polluted soils; however, fewer studies have been conducted in alkaline unpolluted soils. A 4-year field vegetable production experiment was conducted using pig manure (PM) and chicken manure (CM) at five application rates (0, 15, 30, 45, and 60 t ha-1) to elucidate potential risks of HMs in an alkaline unpolluted soil in the Hexi Corridor oasis agricultural area and HM uptake by Chinese cabbage. The results showed that LM application caused a significant build-up of Cu, Zn, Pb, Cd, and Ni content in topsoil by 30.6-99.7%, 11.4-51.7%, 1.4-31.3%, 5.6-44.9%, 14%-40.8%, respectively. The Cd, Cu, Zn could potentially exceed the soil threshold in next 8-65 years after 15-60 t ha-1 LM application. Under LM treatment, the soil DTPA-extractable Cu, Zn, Fe, the acid-extractable fraction of Cu, Zn, Fe, Cd, Ni, and the Oxidable fraction of Cu, Zn, Fe, Mn, Cd, Ni significantly increased, but the DTPA-extractable Pb, Cd, the acid-extractable fraction of Pb, and the reducible fraction of Cd significantly decreased. Cu and Zn could migrate to the deeper soil and relatively increase in DTPA-extracted Cu, Zn were found in 20-40 cm soil depth after LM application. The pH and SOM could influence the bioavailability of HMs in soil. The bioaccumulation factor and transfer factor (TF) values were <1 except Mn (TF > 1). HMs in leaf did not approach the threshold for HM toxicity due to the "dilution effect". Recommend the type of manure was the PM and the annual PM application rate was 30 t ha-1 to ensure a 20-year period of clean production in alkaline unpolluted Fluvo-aqiuc vegetable soils.

Vacuolar Sugar Transporter TMT2 Plays Crucial Roles in Germination and Seedling Development in Arabidopsis.

Vacuolar sugar transporters transport sugar across the tonoplast, are major players in maintaining sugar homeostasis, and therefore play vital roles in plant growth, development, and biomass yield. In this study, we analyzed the physiological roles of the tonoplast monosaccharide transporter 2 (TMT2) in Arabidopsis. In contrast to the wild type (WT) that produced uniform seedlings, the tmt2 mutant produced three types of offspring: un-germinated seeds (UnG), seedlings that cannot form true leaves (tmt2-S), and seedlings that develop normally (tmt2-L). Sucrose, glucose, and fructose can substantially, but not completely, rescue the abnormal phenotypes of the tmt2 mutant. Abnormal cotyledon development, arrested true leaf development, and abnormal development of shoot apical meristem (SAM) were observed in tmt2-S seedlings. Cotyledons from the WT and tmt2-L seedlings restored the growth of tmt2-S seedlings through micrografting. Moreover, exogenous sugar sustained normal growth of tmt2-S seedlings with cotyledon removed. Finally, we found that the TMT2 deficiency resulted in growth defects, most likely via changing auxin signaling, target of rapamycin (TOR) pathways, and cellular nutrients. This study unveiled the essential functions of TMT2 for seed germination and initial seedling development, ensuring cotyledon function and mobilizing sugars from cotyledons to seedlings. It also expanded the current knowledge on sugar metabolism and signaling. These findings have fundamental implications for enhancing plant biomass production or seed yield in future agriculture.

Immunophilin CYN28 is required for accumulation of photosystem II and thylakoid FtsH protease in Chlamydomonas.

Excess light causes severe photodamage to photosystem II (PSII) where the primary charge separation for electron transfer takes place. Dissection of mechanisms underlying the PSII maintenance and repair cycle in green algae promotes the usage of genetic engineering and synthetic biology to improve photosynthesis and biomass production. In this study, we systematically analyzed the high light (HL) responsive immunophilin genes in Chlamydomonas (Chlamydomonas reinhardtii) and identified one chloroplast lumen-localized immunophilin, CYN28, as an essential player in HL tolerance. Lack of CYN28 caused HL hypersensitivity, severely reduced accumulation of PSII supercomplexes and compromised PSII repair in cyn28. The thylakoid FtsH (filamentation temperature-sensitive H) is an essential AAA family metalloprotease involved in the degradation of photodamaged D1 during the PSII repair cycle and was identified as one potential target of CYN28. In the cyn28 mutant, the thylakoid FtsH undergoes inefficient turnover under HL conditions. The CYN28-FtsH1/2 interaction relies on the FtsH N-terminal proline residues and is strengthened particularly under HL. Further analyses demonstrated CYN28 displays peptidyl-prolyl isomerase (PPIase) activity, which is necessary for its physiological function. Taken together, we propose that immunophilin CYN28 participates in PSII maintenance and regulates the homeostasis of FtsH under HL stress via its PPIase activity.

A SPX domain vacuolar transporter links phosphate sensing to homeostasis in Arabidopsis.

Excess phosphate (Pi) is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells. We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level. We tested this hypothesis using Vacuolar Phosphate Transporter 1 (VPT1), a SPX domain-containing vacuolar Pi transporter, as a model. Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules (InsPs) produced at high cellular Pi status. We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1. We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity. Taking together the results from detailed structure-function analyses, our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low, whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole. This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells.

Two magnesium transporters in the chloroplast inner envelope essential for thylakoid biogenesis in Arabidopsis.

Magnesium (Mg2+ ) serves as a cofactor for a number of photosynthetic enzymes in the chloroplast, and is the central atom of the Chl molecule. However, little is known about the molecular mechanism of Mg2+ transport across the chloroplast envelope. Here, we report the functional characterization of two transport proteins in Arabidopsis: Magnesium Release 8 (MGR8) and MGR9, of the ACDP/CNNM family, which is evolutionarily conserved across all lineages of living organisms. Both MGR8 and MGR9 genes were expressed ubiquitously, and their encoded proteins were localized in the inner envelope of chloroplasts. Mutations of MGR8 and MGR9 together, but neither of them alone, resulted in albino ovules and chlorotic seedlings. Further analysis revealed severe defects in thylakoid biogenesis and assembly of photosynthetic complexes in the double mutant. Both MGR8 and MGR9 functionally complemented the growth of the Salmonella typhimurium mutant strain MM281, which lacks Mg2+ uptake capacity. The embryonic and early seedling defects of the mgr8/mgr9 double mutant were rescued by the expression of MGR9 under the embryo-specific ABI3 promoter. The partially rescued mutant plants were hypersensitive to Mg2+ deficient conditions and contained less Mg2+ in their chloroplasts than wild-type plants. Taken together, we conclude that MGR8 and MGR9 serve as Mg2+ transporters and are responsible for chloroplast Mg2+ uptake.

Four plasma membrane-localized MGR transporters mediate xylem Mg2+ loading for root-to-shoot Mg2+ translocation in Arabidopsis.

Magnesium (Mg2+), an essential structural component of chlorophyll, is absorbed from the soil by roots and transported to shoots to support photosynthesis in plants. However, the molecular mechanisms underlying root-to-shoot Mg2+ translocation remain largely unknown. We describe here the identification of four plasma membrane (PM)-localized transporters, named Mg2+ release transporters (MGRs), that are critical for root-to-shoot Mg transport in Arabidopsis. Functional complementation assays in a Mg2+-uptake-deficient bacterial strain confirmed that these MGRs conduct Mg2+ transport. PM-localized MGRs (MGR4, MGR5, MGR6, and MGR7) were expressed primarily in root stellar cells and participated in the xylem loading step of the long-distance Mg2+ transport process. In particular, MGR4 and MGR6 played a major role in shoot Mg homeostasis, as their loss-of-function mutants were hypersensitive to low Mg2+ but tolerant to high Mg2+ conditions. Reciprocal grafting analysis further demonstrated that MGR4 functions in the root to determine shoot Mg2+ accumulation and physiological phenotypes caused by both low- and high-Mg2+ stress. Taken together, our study has identified the long-sought transporters responsible for root-to-shoot Mg2+ translocation in plants.

Conserved mechanism for vacuolar magnesium sequestration in yeast and plant cells.

Magnesium (Mg2+) is an essential nutrient for all life forms. In fungal and plant cells, the majority of Mg2+ is stored in the vacuole but mechanisms for Mg2+ transport into the vacuolar store are not fully understood. Here we demonstrate that members of ancient conserved domain proteins (ACDPs) from Saccharomyces cerevisiae and Arabidopsis thaliana function in vacuolar Mg2+ sequestration that enables plant and yeast cells to cope with high levels of external Mg2+. We show that the yeast genome (as well as other fungal genomes) harbour a single ACDP homologue, referred to as MAM3, that functions specifically in vacuolar Mg2+ accumulation and is essential for tolerance to high Mg. In parallel, vacuolar ACDP homologues were identified from Arabidopsis and shown to complement the yeast mutant mam3Δ. An Arabidopsis mutant lacking one of the vacuolar ACDP homologues displayed hypersensitivity to high-Mg conditions and accumulated less Mg in the vacuole compared with the wild type. Taken together, our results suggest that conserved transporters mediate vacuolar Mg2+ sequestration in fungal and plant cells to maintain cellular Mg2+ homeostasis in response to fluctuating Mg2+ levels in the environment.

Structural variation of mitochondrial genomes sheds light on evolutionary history of soybeans.

The architecture and genetic diversity of mitogenome (mtDNA) are largely unknown in cultivated soybean (Glycine max), which is domesticated from the wild progenitor, Glycine soja, 5000 years ago. Here, we de novo assembled the mitogenome of the cultivar 'Williams 82' (Wm82_mtDNA) with Illumina PE300 deep sequencing data, and verified it with polymerase chain reaction (PCR) and Southern blot analyses. Wm82_mtDNA maps as two autonomous circular chromosomes (370 871-bp Chr-m1 and 62 661-bp Chr-m2). Its structure is extensively divergent from that of the mono-chromosomal mitogenome reported in the landrace 'Aiganhuang' (AGH_mtDNA). Synteny analysis showed that the structural variations (SVs) between two genomes are mainly attributed to ectopic and illegitimate recombination. Moreover, Wm82_mtDNA and AGH_mtDNA each possess six and four specific regions, which are absent in their counterparts and likely result from differential sequence-loss events. Mitogenome SV was further studied in 39 wild and 182 cultivated soybean accessions distributed world-widely with PCR/Southern analyses or a comparable in silico analysis. The results classified both wild and cultivated soybeans into five cytoplasmic groups, named as GSa-GSe and G1-G5; 'Williams 82' and 'Aiganhuang' belong to G1 and G5, respectively. Notably, except for members in GSe and G5, all accessions carry a bi-chromosomal mitogenome with a common Chr-m2. Phylogenetic analyses based on mtDNA structures and chloroplast gene sequences both inferred that G1-G3, representing >90% of cultigens, likely inherited cytoplasm from the ancestor of domestic soybean, while G4 and G5 likely inherited cytoplasm from wild soybeans carrying GSa- and GSe-like cytoplasm through interspecific hybridization, offering new insights into soybean cultivation history.

Functional Relationship of Arabidopsis AOXs and PTOX Revealed via Transgenic Analysis.

Alternative oxidase (AOX) and plastid terminal oxidase (PTOX) are terminal oxidases of electron transfer in mitochondria and chloroplasts, respectively. Here, taking advantage of the variegation phenotype of the Arabidopsis PTOX deficient mutant (im), we examined the functional relationship between PTOX and its five distantly related homologs (AOX1a, 1b, 1c, 1d, and AOX2). When engineered into chloroplasts, AOX1b, 1c, 1d, and AOX2 rescued the im defect, while AOX1a partially suppressed the mutant phenotype, indicating that AOXs could function as PQH2 oxidases. When the full length AOXs were overexpressed in im, only AOX1b and AOX2 rescued its variegation phenotype. In vivo fluorescence analysis of GFP-tagged AOXs and subcellular fractionation assays showed that AOX1b and AOX2 could partially enter chloroplasts while AOX1c and AOX1d were exclusively present in mitochondria. Surprisingly, the subcellular fractionation, but not the fluorescence analysis of GFP-tagged AOX1a, revealed that a small portion of AOX1a could sort into chloroplasts. We further fused and expressed the targeting peptides of AOXs with the mature form of PTOX in im individually; and found that targeting peptides of AOX1a, AOX1b, and AOX2, but not that of AOX1c or AOX1d, could direct PTOX into chloroplasts. It demonstrated that chloroplast-localized AOXs, but not mitochondria-localized AOXs, can functionally compensate for the PTOX deficiency in chloroplasts, providing a direct evidence for the functional relevance of AOX and PTOX, shedding light on the interaction between mitochondria and chloroplasts and the complex mechanisms of protein dual targeting in plant cells.

Identification of interacting proteins of Arabidopsis cyclophilin38 (AtCYP38) via multiple screening approaches reveals its possible broad functions in chloroplasts.

AtCYP38, a thylakoid lumen localized immunophilin, is found to be essential for photosystem II assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen. To identify potential interacting proteins of AtCYP38, we first adopted ATTED-II and STRING web-tools, and found 12 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with different domain of AtCYP38, and five thylakoid lumen-localized targets were identified. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we generated a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumenal fractions of thylakoid membrane proteins, and we obtained six thylakoid membrane proteins and nine thylakoid lumenal proteins as interacting proteins of AtCYP38. The interactions between AtCYP38 and several potential targets were further confirmed via pull-down and co-immunoprecipitation assays. Together, a couple of new potential candidate interacting proteins of AtCYP38 were identified, and the results will lay a foundation for unveiling the regulatory mechanisms in photosynthesis by AtCYP38.

Characterization of the complete chloroplast genome of Murraya exotica (Rutaceae) from Yunnan Province, China.

Murraya exotica L. (Rutaceae) has important horticultural and medicinal values. Here, we reported the complete chloroplast (cp) genome of M. exotica using the next-generation sequencing method. The cp genome is 160,179 bp in length, including a large single-copy region (LSC, 87,726 bp), a small single-copy region (SSC, 18,465 bp), and a pair of inverted repeats (IR) regions 26,994 bp. A maximum-likelihood phylogenomic analysis showed that M. exotica was sister to Murraya paniculate. These findings will provide useful information for further investigation of cp genome evolution in Murraya.

Basic Helix-Loop-Helix (bHLH) Transcription Factors Regulate a Wide Range of Functions in Arabidopsis.

The basic helix-loop-helix (bHLH) transcription factor family is one of the largest transcription factor gene families in Arabidopsis thaliana, and contains a bHLH motif that is highly conserved throughout eukaryotic organisms. Members of this family have two conserved motifs, a basic DNA binding region and a helix-loop-helix (HLH) region. These proteins containing bHLH domain usually act as homo- or heterodimers to regulate the expression of their target genes, which are involved in many physiological processes and have a broad range of functions in biosynthesis, metabolism and transduction of plant hormones. Although there are a number of articles on different aspects to provide detailed information on this family in plants, an overall summary is not available. In this review, we summarize various aspects of related studies that provide an overview of insights into the pleiotropic regulatory roles of these transcription factors in plant growth and development, stress response, biochemical functions and the web of signaling networks. We then provide an overview of the functional profile of the bHLH family and the regulatory mechanisms of other proteins.

A single nucleotide polymorphism in an R2R3 MYB transcription factor gene triggers the male sterility in soybean ms6 (Ames1).

KEY MESSAGE: Identification and functional analysis of the male sterile gene MS6 in Glycine max. Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.

Diverged Early From CtpB and CtpC, CtpA Has Evolved to Process D1 Precursor in Oxygenic Photosynthetic Organisms.

C-terminal peptidase (Ctp) cleaves the C-terminal extension of the D1 precursor (pD1) to form mature D1. Among the three homologs CtpA, CtpB, and CtpC in photosynthetic organisms only the first is capable of processing pD1 while the roles of CtpB and CtpC remain elusive. Phylogenetic analysis of Ctps from photosynthetic organisms revealed that CtpA has diverged early from CtpB and CtpC during evolution implying distinct roles for the Ctps. Analysis of Arabidopsis Ctp-deficient mutants revealed that pD1 processing was not affected in atctpb, atctpc, or atctpbatctpc mutants, demonstrating that AtCtpA, not AtCtpB or AtCtpC, is responsible for cleaving the pD1 C-terminal extension. Ectopic expression of CtpAs from Synechococcus elongatus, Chlamydomonas reinhardtii, and Physcomitrella patens in atctpa rescued the lethal phenotype of the mutant indicating that SeCtpA, CrCtpA, and PpCtpA could process pD1 in Arabidopsis. Enzyme activity assays showed that PpCtpA and CrCtpA could convert pD1 into mature D1 in vitro. In contrast, expressing CtpB or CtpC from Arabidopsis, C. reinhardtii, or P. patens in atctpa did not rescue its D1 maturation deficiency, and enzyme activity assays also showed that neither CtpB nor CtpC could process pD1 in vitro. Taken together, we conclude that the function of pD1 processing by CtpA is conserved in photosynthetic organisms. It is possible that among other factors CtpA developed this function to initiate the formation of the oxygenic D1/D2 type PSII complex during evolution whereas CtpB or CtpC have other roles that are still unclear.

A Golgi-localized manganese transporter functions in pollen tube tip growth to control male fertility in Arabidopsis.

Manganese (Mn) serves as an essential cofactor for many enzymes in various compartments of a plant cell. Allocation of Mn among various organelles thus plays a central role in Mn homeostasis to support metabolic processes. We report the identification of a Golgi-localized Mn transporter (named PML3) that is essential for rapid cell elongation in young tissues such as emerging leaves and the pollen tube. In particular, the pollen tube defect in the pml3 loss-of-function mutant caused severe reduction in seed yield, a critical agronomic trait. Further analysis suggested that a loss of pectin deposition in the pollen tube might cause the pollen tube to burst and slow its elongation, leading to decreased male fertility. As the Golgi apparatus serves as the major hub for biosynthesis and modification of cell-wall components, PML3 may function in Mn homeostasis of this organelle, thereby controlling metabolic and/or trafficking processes required for pectin deposition in rapidly elongating cells.

Conserved Residues in the C-Terminal Domain Affect the Structure and Function of CYP38 in Arabidopsis.

Arabidopsis cyclophilin38 (CYP38) is a thylakoid lumen protein critial for PSII assembly and maintenance, and its C-terminal region serves as the target binding domain. We hypothesized that four conserved residues (R290, F294, Q372, and F374) in the C-terminal domain are critical for the structure and function of CYP38. In yeast two-hybrid and protein pull-down assays, CYP38s with single-sited mutations (R290A, F294A, Q372A, or F374A) did not interact with the CP47 E-loop as the wild-type CYP38. In contrast, CYP38 with the R290A/F294A/Q372A/F374A quadruple mutation could bind the CP47 E-loop. Gene transformation analysis showed that the quadruple mutation prevented CYP38 to efficiently complement the mutant phenotype of cyp38. The C-terminal domain half protein with the quadruple mutation, like the wild-type one, could interact with the N-terminal domain or the CP47 E-loop in vitro. The cyp38 plants expressing CYP38 with the quadruple mutation showed a similar BN-PAGE profile as cyp38, but distinct from the wild type. The CYP38 protein with the quadruple mutation associated with the thylakoid membrane less efficiently than the wild-type CYP38. We concluded that these four conserved residues are indispensable as changes of all these residues together resulted in a subtle conformational change of CYP38 and reduced its intramolecular N-C interaction and the ability to associate with the thylakoid membrane, thus impairing its function in chloroplast.